The enormous production potential of buffaloes has never been accomplished due to various reproductive insufficiencies. Among them, post-partum anoestrus, a multifactorial disorder, is predominant but any genetic association is yet to be established. This study focused to identify novel polymorphisms in heat-shock protein 70 (HSP70) gene and its possible association with post-partum anoestrus in Murrah buffaloes. A 579-bp fragment from 5' untranslated region of HSP70 gene was amplified by polymerase chain reaction from blood genomic DNA of 614 animals maintained under similar management conditions. In phase-I experiment, custom sequencing and restriction enzyme (RE) digestion of the amplified fragment were performed in 40 buffaloes with similar post-partum oestrous conditions over previous consecutive three or more gestations-20 animals each showing post-partum anoestrus (>120 days after parturition) and normal cyclicity (<65 days after parturition). While in phase-II experiment, herd screening by RE analysis was performed in remaining 574 animals. Four transversions at T-75G, C+31G, T+38G and C+97A and three transition mutations at T-153C, T+33C and A+44G positions were observed. Polymorphism at T+38G site revealed significant (p < .05) variation, where homozygous G was present only in post-partum anoestrous animals while nucleotide T was present randomly in both groups of phase-I animals whereas phase-II experiments revealed homozygous G in 55 animals. Regression analysis in relation to average post-partum interval against genotypic frequencies at T+38G also depicted significant association. HSP70 gene polymorphism at T+38G position can therefore be used as genetic marker for excluding probable post-partum anoestrous buffaloes from herd for breeding programmes.