Postpartum anestrus (PPA) condition or long PPA interval (PPAI) (>90 days) is a reproductive problem in buffaloes. Both genetic and non-genetic factors contribute to the variation in PPAI. Identifying the genetic markers associated with PPA will help in the marker-based selection of buffaloes against PPA, thereby PPA incidence can be reduced. However, such genetic markers associated with PPA are scanty. Therefore, the present study was targeted to identify the association of SNPs in the gonadotropin inhibitory hormone (GnIH/NPVF) gene with PPAI in Indian Murrah buffaloes. GnIH/NPVF is a neuropeptide that inhibits gonadotropin-releasing hormone (GnRH) and gonadotropin secretion. Sequencing of the GnIH/NPVF gene amplified from a pooled DNA sample of 10 PPA and 10 healthy postpartum cyclic controls revealed 8 SNPs in the putative promoter and 5’-UTR regions. Transcription factor binding analysis identified that the allele A of the SNP (c.-1001A>G) at the 5’UTR region could promote the binding of FoxO_CS transcription factor, an inhibitor of gonadotropin release. Association analysis between the genotypes of this SNP and PPAI of 66 extreme PPA buffaloes and 83 extreme controls (PPA< 90 days) identified its significant (P<0.0093) association with PPAI in this selected buffalo population sample. Particularly, the G-allele of this SNP appeared to reduce 30.33±11.51 PPAI days in Murrah buffaloes. In conclusion, the SNP c.-1001A>G in the GnIH/NPVF gene could be a potential genetic marker associated with PPA in Murrah buffaloes, and this association needs further validation in large population samples before its consideration for marker-assisted selection programs against PPA in buffaloes.