The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds.