The present study was undertaken with 204 buffaloes during June, 2016 to July, 2017 from different locations viz., Saraswathi Krishi Vigyan Kendra, Karur district, Tamil Nadu; Post Graduate Research Institute in Animal Sciences (TANUVAS), Katupakkam, Tamil Nadu; Central Cattle Breeding Farm, Alamadhi, Chennai, Tamil Nadu; Buffalo Research Station, Venkataramanna Gudem, S.V.V.U, West Godavari District, Andhra Pradesh and Farmers herd in Namakkal, Tamil Nadu to determine the genetic variation. The animals were genotyped by Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP). In the PCR reaction the primers used were 5’ AGC ATC TAT ACA GCA TGA AGT G 3’ and 5’ GTG TAC TAA GCT GAC AAA GTC TTG 3’. An 870 bp long ER-α (exon 13) gene PCR product was genotyped for polymorphic pattern using MboI restriction enzymes. The PCR-RFLP of ER-α gene (exon 13) showed 614 and 256 bp long digested PCR products in all the tested samples. Monomorphic pattern as AA genotype for all the tested animals indicates the fixation of this allele in Murrah buffaloes.