Staphylococcus aureus is arguably the most important pathogen involved in bovine mastitis. The aim of this study was to determine the virulence gene profiles of 124 Staph. aureus isolates from subclinical mastitis in cows in eastern Poland. The presence of 30 virulence genes encoding adhesins, proteases and superantigenic toxins was investigated by PCR. The 17 different combinations of adhesin genes were identified. Occurrence of eno (91·1%) and fib (82·3%) genes was found to be common. The frequency of other adhesion genes fnbA, fnbB, ebps were 14·5, 50, 25%, respectively, and for cna and bbp were 1·6%. The etA and etD genes, encoding exfoliative toxins, were present in genomes of 5·6 and 8·9% isolates, respectively. The splA and sspA, encoding serine protease, were detected in above 90% isolates. The most frequent enterotoxin genes were sei (21%), sem (19·4%), sen (19·4%), seg (18·5%) and seo (13·7%). The tst gene was harboured by 2·4% isolates. The 19 combinations of the superantigenic toxin genes were obtained and found in 35·5% of isolates. Three of them (seg, sei, sem, sen, seo; sec, seg, sei, sem, sen, seo and seg, sei, sem, sen) were the most frequent and found in 16·1% of the isolates. The most common virulotype, present in 17·7% of the isolates, was fib, eno, fnbB, splA, splE, sspA. The results indicate the variation in the presence of virulence genes in Staph. aureus isolates and considerable diversity of isolates that are able to cause mastitis in cows.