Abstract: Genotypes of 72 Russian Black pied and 98 Red pied cows were determined for PRL-Rsa I loci by Restriction Fragment Length Polymorphism analysis (PCR-RFLP) of amplified DNA. This technique was used to determine the PRL-Rsa I allelic frequency in Black pied and Red pied dairy herds. Estimated gene frequencies were 0.71, 0.79 for the A and 0.29, 0.21 for the B alleles PRL-Rsa I for Black pied and Red pied breeds, respectively. There were no difference in the frequency of A and B alleles of Prolactin gene. A significant association between the PRL-Rsa I and milk yield, fat percentage is shown for two breeds.