Staphylococcus aureusone of the most important pathogens involved in bovine mastitis. The aim of this study was to determine the virulence and resistance gene profiles of 26 Staph. aureusisolatedfrom bovine mastitis milk in A-Amarah City, Maysan, Iraq. The presence of 4 virulence genes (tst,sirB, icaA, and hlg) that encoding for Toxic shock syndrome toxin, Siderophore compound transporter permease protein, Polysaccharide Intracellular adhesion, and Gama-hemolysin respectively, and 2 resistant genes (mecAand femA) that encoding for Methicillin resistance wereinvestigated by PCR. The occurrence of sirBand femAgenes (100%) was found to be common. The frequency of other genes was three (11.53%) of isolate expressed the mecAgene, four (15.38%) hlggene, seven (26.92%) tstgene and 24 (92.30%) had icaA. The results indicate the variation in the presence of virulence genes in Staphylococcus aureus isolates that are able to cause mastitis in cowsand PCR assays canbe used as rapid diagnostic tools to detect the virulence factors and antimicrobial susceptibility testing of S. aureusthat help in the detection of the severity of the infection.