Buffalo milk is economically important, and an understanding of the genetic basis of milk production traits may facilitate buffalo breeding for this purpose. In this study, polymerase chain reaction-single strand conformation polymorphism was used to analyze polymorphisms in prolactin receptor (PRLR) exons 3, 5, 7, and 10 in Murrah, Nili-Ravi, and crossbred buffaloes and Holstein cows. A site in PRLR exon 3 exhibited variation in all three buffalo breeds and cows, and the three genotypes GG, GT, and TT were detected. In PRLR exon 5, nucleotide differences were detected between buffalo and Holstein dairy cow populations; the genotypes AA, AG, and GG were observed. In PRLR exon 7, polymorphisms were not detected. In PRLR exon 10, at 9637 bp, the TT, TC, and TT genotypes were observed. At the three polymorphic loci, Hardy–Weinberg equilibrium was reached in the crossbred buffalo population for exons 3 and 5 and in all three buffalo populations for exon 10. In an analysis of the associations of the three polymorphisms with milk production traits, only PRLR exon 10 in Murrah buffaloes was associated with milk yield, with a significant difference between the TT and CC genotypes.