<?xml version="1.0"?>
<edgarSubmission>

    <schemaVersion>X0708</schemaVersion>

    <submissionType>D/A</submissionType>

    <testOrLive>LIVE</testOrLive>

    <primaryIssuer>
        <cik>0001301204</cik>
        <entityName>Chalkstream Investment Fund, L.P.</entityName>
        <issuerAddress>
            <street1>444 MADISON AVENUE, 22ND FLOOR</street1>
            <city>NEW YORK</city>
            <stateOrCountry>NY</stateOrCountry>
            <stateOrCountryDescription>NEW YORK</stateOrCountryDescription>
            <zipCode>10022</zipCode>
        </issuerAddress>
        <issuerPhoneNumber>(212) 707-6100</issuerPhoneNumber>
        <jurisdictionOfInc>DELAWARE</jurisdictionOfInc>
        <edgarPreviousNameList>
            <previousName>Chalkstream Investment Fund L P</previousName>
        </edgarPreviousNameList>
        <entityType>Limited Partnership</entityType>
        <yearOfInc>
            <overFiveYears>true</overFiveYears>
        </yearOfInc>
    </primaryIssuer>

    <relatedPersonsList>
        <relatedPersonInfo>
            <relatedPersonName>
                <firstName>-</firstName>
                <lastName>Chalkstream GP, L.L.C.</lastName>
            </relatedPersonName>
            <relatedPersonAddress>
                <street1>1602 Windy Ridge Drive</street1>
                <city>Brentwood</city>
                <stateOrCountry>TN</stateOrCountry>
                <stateOrCountryDescription>TENNESSEE</stateOrCountryDescription>
                <zipCode>37027</zipCode>
            </relatedPersonAddress>
            <relatedPersonRelationshipList>
                <relationship>Promoter</relationship>
            </relatedPersonRelationshipList>
            <relationshipClarification>The General Partner of the Issuer.</relationshipClarification>
        </relatedPersonInfo>
        <relatedPersonInfo>
            <relatedPersonName>
                <firstName>Andrew</firstName>
                <lastName>Weinman</lastName>
            </relatedPersonName>
            <relatedPersonAddress>
                <street1>c/o Chalkstream Capital Group, L.P.</street1>
                <street2>1602 Windy Ridge Drive</street2>
                <city>Brentwood</city>
                <stateOrCountry>TN</stateOrCountry>
                <stateOrCountryDescription>TENNESSEE</stateOrCountryDescription>
                <zipCode>37027</zipCode>
            </relatedPersonAddress>
            <relatedPersonRelationshipList>
                <relationship>Executive Officer</relationship>
            </relatedPersonRelationshipList>
            <relationshipClarification>Managing Principal of Chalkstream GP L.L.C., the General Partner of the Issuer.</relationshipClarification>
        </relatedPersonInfo>
    </relatedPersonsList>

    <offeringData>
        <industryGroup>
            <industryGroupType>Pooled Investment Fund</industryGroupType>
            <investmentFundInfo>
                <investmentFundType>Hedge Fund</investmentFundType>
                <is40Act>false</is40Act>
            </investmentFundInfo>
        </industryGroup>
        <issuerSize>
            <aggregateNetAssetValueRange>Decline to Disclose</aggregateNetAssetValueRange>
        </issuerSize>
        <federalExemptionsExclusions>
            <item>06b</item>
            <item>3C</item>
            <item>3C.7</item>
        </federalExemptionsExclusions>
        <typeOfFiling>
            <newOrAmendment>
                <isAmendment>true</isAmendment>
                <previousAccessionNumber>0001301204-25-000003</previousAccessionNumber>
            </newOrAmendment>
            <dateOfFirstSale>
                <value>2004-03-01</value>
            </dateOfFirstSale>
        </typeOfFiling>
        <durationOfOffering>
            <moreThanOneYear>true</moreThanOneYear>
        </durationOfOffering>
        <typesOfSecuritiesOffered>
            <isPooledInvestmentFundType>true</isPooledInvestmentFundType>
            <isOtherType>true</isOtherType>
            <descriptionOfOtherType>Limited Partnership Interests</descriptionOfOtherType>
        </typesOfSecuritiesOffered>
        <businessCombinationTransaction>
            <isBusinessCombinationTransaction>false</isBusinessCombinationTransaction>
            <clarificationOfResponse></clarificationOfResponse>
        </businessCombinationTransaction>
        <minimumInvestmentAccepted>0</minimumInvestmentAccepted>
        <salesCompensationList>
            <recipient>
                <recipientName>-</recipientName>
                <recipientCRDNumber>None</recipientCRDNumber>
                <associatedBDName>IVP Capital, LLC</associatedBDName>
                <associatedBDCRDNumber>104197</associatedBDCRDNumber>
                <recipientAddress>
                    <street1>337 NOYAC PATH</street1>
                    <city>WATER MILL</city>
                    <stateOrCountry>NY</stateOrCountry>
                    <stateOrCountryDescription>NEW YORK</stateOrCountryDescription>
                    <zipCode>11976</zipCode>
                </recipientAddress>
                <statesOfSolicitationList>
                    <state>CA</state>
                    <description>CALIFORNIA</description>
                    <state>CT</state>
                    <description>CONNECTICUT</description>
                    <state>DC</state>
                    <description>DISTRICT OF COLUMBIA</description>
                    <state>FL</state>
                    <description>FLORIDA</description>
                    <state>IL</state>
                    <description>ILLINOIS</description>
                    <state>MA</state>
                    <description>MASSACHUSETTS</description>
                    <state>MT</state>
                    <description>MONTANA</description>
                    <state>NY</state>
                    <description>NEW YORK</description>
                    <state>NC</state>
                    <description>NORTH CAROLINA</description>
                    <state>PA</state>
                    <description>PENNSYLVANIA</description>
                    <state>TX</state>
                    <description>TEXAS</description>
                    <state>VA</state>
                    <description>VIRGINIA</description>
                </statesOfSolicitationList>
                <foreignSolicitation>true</foreignSolicitation>
            </recipient>
            <recipient>
                <recipientName>-</recipientName>
                <recipientCRDNumber>None</recipientCRDNumber>
                <associatedBDName>Gallatin Capital LLC</associatedBDName>
                <associatedBDCRDNumber>124738</associatedBDCRDNumber>
                <recipientAddress>
                    <street1>444 MADISON AVENUE, 29TH FLOOR</street1>
                    <city>NEW YORK</city>
                    <stateOrCountry>NY</stateOrCountry>
                    <stateOrCountryDescription>NEW YORK</stateOrCountryDescription>
                    <zipCode>10022</zipCode>
                </recipientAddress>
                <statesOfSolicitationList>
                    <state>AL</state>
                    <description>ALABAMA</description>
                    <state>AK</state>
                    <description>ALASKA</description>
                    <state>AZ</state>
                    <description>ARIZONA</description>
                    <state>CA</state>
                    <description>CALIFORNIA</description>
                    <state>CO</state>
                    <description>COLORADO</description>
                    <state>CT</state>
                    <description>CONNECTICUT</description>
                    <state>DC</state>
                    <description>DISTRICT OF COLUMBIA</description>
                    <state>FL</state>
                    <description>FLORIDA</description>
                    <state>GA</state>
                    <description>GEORGIA</description>
                    <state>IA</state>
                    <description>IOWA</description>
                    <state>KY</state>
                    <description>KENTUCKY</description>
                    <state>ME</state>
                    <description>MAINE</description>
                    <state>MD</state>
                    <description>MARYLAND</description>
                    <state>MA</state>
                    <description>MASSACHUSETTS</description>
                    <state>MI</state>
                    <description>MICHIGAN</description>
                    <state>MO</state>
                    <description>MISSOURI</description>
                    <state>NE</state>
                    <description>NEBRASKA</description>
                    <state>NH</state>
                    <description>NEW HAMPSHIRE</description>
                    <state>NJ</state>
                    <description>NEW JERSEY</description>
                    <state>NY</state>
                    <description>NEW YORK</description>
                    <state>NC</state>
                    <description>NORTH CAROLINA</description>
                    <state>OH</state>
                    <description>OHIO</description>
                    <state>PA</state>
                    <description>PENNSYLVANIA</description>
                    <state>RI</state>
                    <description>RHODE ISLAND</description>
                    <state>SC</state>
                    <description>SOUTH CAROLINA</description>
                    <state>SD</state>
                    <description>SOUTH DAKOTA</description>
                    <state>TN</state>
                    <description>TENNESSEE</description>
                    <state>TX</state>
                    <description>TEXAS</description>
                    <state>UT</state>
                    <description>UTAH</description>
                    <state>VT</state>
                    <description>VERMONT</description>
                    <state>VA</state>
                    <description>VIRGINIA</description>
                    <state>WA</state>
                    <description>WASHINGTON</description>
                    <state>WV</state>
                    <description>WEST VIRGINIA</description>
                    <state>WI</state>
                    <description>WISCONSIN</description>
                    <state>WY</state>
                    <description>WYOMING</description>
                </statesOfSolicitationList>
                <foreignSolicitation>true</foreignSolicitation>
            </recipient>
        </salesCompensationList>
        <offeringSalesAmounts>
            <totalOfferingAmount>Indefinite</totalOfferingAmount>
            <totalAmountSold>751557986</totalAmountSold>
            <totalRemaining>Indefinite</totalRemaining>
            <clarificationOfResponse></clarificationOfResponse>
        </offeringSalesAmounts>
        <investors>
            <hasNonAccreditedInvestors>false</hasNonAccreditedInvestors>
            <totalNumberAlreadyInvested>163</totalNumberAlreadyInvested>
        </investors>
        <salesCommissionsFindersFees>
            <salesCommissions>
                <dollarAmount>133762</dollarAmount>
            </salesCommissions>
            <findersFees>
                <dollarAmount>0</dollarAmount>
            </findersFees>
            <clarificationOfResponse>Clarification of Item 12.  IVP Capital, LLC and Gallatin Capital LLC are no longer in a marketing agreement with the Issuer, in connection with sales of securities in the offering.</clarificationOfResponse>
        </salesCommissionsFindersFees>
        <useOfProceeds>
            <grossProceedsUsed>
                <dollarAmount>0</dollarAmount>
            </grossProceedsUsed>
            <clarificationOfResponse>Customary advisory fees are paid to the Management Company as provided for in the Issuer's offering materials.</clarificationOfResponse>
        </useOfProceeds>
        <signatureBlock>
            <authorizedRepresentative>false</authorizedRepresentative>
            <signature>
                <issuerName>Chalkstream Investment Fund, L.P.</issuerName>
                <signatureName>Andrew Weinman</signatureName>
                <nameOfSigner>Andrew Weinman</nameOfSigner>
                <signatureTitle>Managing Principal of the General Partner</signatureTitle>
                <signatureDate>2026-04-09</signatureDate>
            </signature>
        </signatureBlock>
    </offeringData>
</edgarSubmission>
