Melanin is the pigment of skin (coat) and hairs in all mammals. The 954 bp long MC1R gene codes for the production of MC1R or the α-MSH receptors which are present on the melanosomes. These receptors regulate the synthesis of melanin pigments viz., eumelanin or phaeomelanin leading to black and reddish to yellowish pigmentations respectively. PCR-RFLP analysis of 138 bp region of MC1R gene was conducted in two buffalo populations having distinct coat colours; Nagpuri (black) and Purnathadi (whitish). Three genotypes, EDED, EDE* and E*E* were observed at frequencies of 0.86, 0.03 and 0.11 for Nagpuri and 0.74, 0.08 and 0.18 in Purnathadi buffaloes; similarly the observed gene frequencies for ED and E* alleles were 0.87, 0.13 and 0.78, 0.22 in Nagpuri and Purnathadi buffaloes, respectively. DNA-sequencing revealed a transition mutation (A→G) at 118 bp locus confirming polymorphism, however presence in both the population failed to point towards any specific role in body coat colour of buffaloes.