The objective of this study was to investigate polymorphisms both in exons 1 and 3 of prolactin (PRL) gene for milk productivity of Anatolian water buffalo breed in Sivas province in Turkey. Blood samples were collected from 129 male and female water buffaloes and DNA was isolated by using phenol/chloroform method. Samples of DNA were amplified and resulting PCR products were digested with RsaI (for exons 1 and 3) and HaeIII (for exon 1). Allelic polymorphisms were determined by separation of fragments obtained from digested PCR products in 3% agarose gel electrophoresis. AA genotype (HaeIII) and BB genotype (RsaI) of exon 1 and only AA genotype (RsaI) of exon 3 were obtained. No polymorphisms were determined in Anatolian water buffalo breed and all loci were found as monomorphic. It can be stated that Anatolian water buffalo has a higher milk and milk fat yields since BB genotype was obtained.