In this study, prolactin gene polymorphism was investigated in Nili-Ravi buffaloes, Sahiwal and Achai cattle breeds, 100 per group, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Only genotype GG was observed in the case of Nili-Ravi buffaloes. In Sahiwal and Achai cattle, three genotypes were found, AA, AG and GG: the frequency of these genotypes were 72%, 18% and 10% in Sahiwal cattle and 44%, 34% and 22% in Achai cattle, respectively. The frequency of genotype AA was found to be higher in both cattle breeds. Results of chi-square test at P < 0.05 revealed that animals of Achai cattle were in Hardy-Weinberg equilibrium, whereas Sahiwal cattle were found to be deviating.