Staphylococcus aureus is often responsible for a number of diseases in humans and animals due to the production of different virulence factors. Therefore, present study investigated the virulence genes of Staphylococcus aureus isolated from bovine mastitis. Out of 52 isolates of Staphylococcus aureus, all the isolates were found negative for icaA gene whereas 18 (34.61 %) isolates were found positive for icaD gene producing 381 bp amplicon. In detection of hemolysine gene hlA and hlB, out of 52 isolates of Staphylococcus aureus, 15 (28.84 %) and 11 (21.15 %) isolates were found positive for presence of hlA and hlB gene, respectively. Out of 52 isolates of Staphylococcus aureus, 43 (82.69 %) isolates were found positive for coa gene in which single amplicons of 550, 680 and 850 bp were detected among 19 (36.53 %), 11 (21.15 %) and 13 (25.00%) isolates, respectively. In the detection of X-region of Protein A in spa gene by PCR, 18 (34.61 %) isolates isolates of Staphylococcus aureus out of 52 were found positive in which single amplicons of 180, 206 and 270 bp were detected among 6 (11.53%), 4 (07.69%) and 8 (15.38%) isolates, respectively indicative of polymorphisms of spa gene. These results revealed that mastitis associated Staphylococcus aureus among bovines is able to accumulate different virulence factors making the treatment of infections difficult.