In this study, polymorphism of prolactin gene was analyzed as a candidate gene responsible for variation and genetic trends in milk yield and composition traits. Genomic DNAs were extracted from 268 semen samples belonged to Iranian Holstein bulls. Genotyping for the prolactin gene using PCRRFLP technique and RsaI restriction endonuclease showed a mutation in 294-bp fragment located in exon 4. The frequencies of A and G alleles were 0.069 and 0.931, respectively. The allelic substitution effect was significant for milk and protein yield (p < 0.05). The G allele was unfavorable for milk and
protein yield. Genetic trends for all analyzed traits were significant (p < 0.01) and that was progressive for milk, fat and protein yield, but diminishing for fat and protein percent. The effects of prolactin SNP on genetic trends and the difference between genetic trends produced by A and G alleles were not
significant for all studied traits.