Prolactin (PRL) plays an important role in regulating mammary gland development, secreting milk, and expressing milk protein genes; making it a potential genetic marker and a candidate gene for production traits in dairy animals. The aim of the study was to determine by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method the gene and genotype frequencies of PRL gene in native East Anatolian Red (EAR) cattle, which are raised as a genetic resource in Turkey. PCR-RFLP analysis involved the use of the RsaI restriction enzyme. Three patterns of fragments were obtained. The AA, AG, and GG genotype frequencies were 0.07, 0.34, and 0.59 in the cattle population, respectively. For Prolactin-RsaI (PRL-RsaI) polymorphism, the population was in Hardy-Weinberg equilibrium. Heterozygosity was found at a medium rate as 0.338 and the calculated FIS value was 0.072.