Milk protein genes expression in cows' mammary epithelial cells is regulated mostly by the action of prolactin mediated through the STAT5A transcription factor. The STAT5A gene is a potential quantitative trait locus (QTL) and genetic marker of production traits in dairy cattle. The sequence of the bovine STAT5A gene was analysed in this study to investigate if mutations in this sequence might be responsible for quantitative variations in milk yield and composition. Ten PCR fragments representing most important functional domains of STAT5A were screened for polymorphism. Using the SSCP method a new SNP (A/G) was found, located in intron 9 at position 9501 (GenBank AJ237937). The frequencies of alleles were estimated in 186 Black-and-White cows (0.52 and 0.48 for A and G, respectively) and in 138 Jersey cows (0.58 and 0.42 for A and G, respectively). For Black-and-White cows with different STAT5A genotypes no significant associations between STAT5A genotypes and milk performance traits were found. Statistically significant differences in the first and second lactations for milk yield, fat and protein content were found in Jersey cows. Cows with the GG genotype showed the highest milk yield, while cows with genotypes AA and AG showed higher protein contents when compared to cows with the GG genotype. Interestingly, cows with genotype AG showed significantly higher protein yields in comparison to cows with the AA genotype. For fat content, cows with genotype AA showed the highest level of this trait in the 1st and 2nd lactation. Further studies are necessary to evaluate an allele substitution effect in the population of sib-families of STAT5A heterozygous bulls.