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The study was envisaged to investigate the polymorphism, expression pattern and association of Heat Shock Protein 70 (HSP 70) polymorphs with thermotolerance traits on fifty Murrah buffaloes using advanced molecular technique. SSCP banding pattern of 503bp fragment of HSP 70 gene revealed three genotypes AA, AB, BB comprised of A and B alleles in studied Murrah buffalo population. The respective genotype frequencies and allelic frequencies were estimated. RT-PCR studied of HSP 70 gene of Murrah buffaloes indicated that its expression was significantly higher (p 0.01) in summer season while it was nearly halved in winter season as compared to spring season. Least square analysis of data showed that AB genotype had significantly lowest (p 0.05) respiration and pulse rate compared to AA and BB genotype. Hence, rearing of Murrah buffalo with AB genotype is more preferable than AA and BB genotype for heat stress management.