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A number of proteins are expressed specifically in X- and Y-chromosome bearing spermatozoa. The Sry protein is known to be expressed only in Y-chromosome bearing spermatozoa and also in other tissues like brain, kidney and adrenal gland of adult males. Although expression of buffalo Sry protein has not been reported earlier, the recombinant Sry can be used to understand its role in the tissues. Further, protein based test is needed for assessment of sorted semen. The present study was undertaken to clone and express the buffalo SRY gene. The recombinant protein was expressed as inclusion bodies. The expressed fusion protein showed a mol wt of about 27.19 kDa. However, Western blotting with sperm cell lysate showed a band at about 31 kDa. The HMG-box of Sry protein had shown 6 amino acid substitutions as compared to the Bos taurus and these would probably make buffalo Sry functionally more active compared to its closely related species. The recombinant Sry could be used to raise monoclonal antibodies for labelling the Y-chromosome bearing spermatozoa, which can be used for assessing the enrichment of Y-chromosome bearing spermatozoa in sex sorted semen.