Prolactin plays an important role in mammary gland development, milk secretion, and expression of milk protein genes. thus the gene is a potential quantitative trait locus and genetic marker of production traits in dairy animals. Therefore, present study aimed to screen buffalo herd maintained at National Dairy Research Institute, Karnal for the genetic polymorphism of the PRL gene using PCR–RFLP analysis for future use of genetic variants as markers for animal selection. Genomic DNA was isolated from 150 Murrah buffaloes. PCR was set up to amplify exon 3 of prolactin locus using specific primers. PCR-RFLP analysis was carried out by digestion of 156 bp amplicon with Rsa I restriction enzyme. The animals included in the study exhibited two genotypes AB and BB with frequencies of 0.97 and 0.03 with allelic frequencies of A and B alleles respectively, indicative of very low polymorphic content. DNA sequencing revealed three variations at three nucleotide position, G5443T both in exon 3 region out of which C5396G resulting in amino acid substitution from leucine to valine. Third change A5463G was in intron 3 region. Such a methodology of true genotyping of the locus may further be utilized in developing specific breeding programmes for genetic variants for their future use as genetic markers for animal selection. 1337018272.php