Staphylococcus aureus is the most frequently bacterial pathogen causing clinical and subclinical mastitis in cattle. In this research the mastitic cases (103) were classified into clinical and subclinical cases .(47,56) respectively. All milk samples collected from infected cows were subjected to bacteriological examination and molecular characterization of some Staphylococcus aureus isolates. Staphylococcus aureus was isolated from clinical and subclinical mastitic cows in an incidence of (50%,17%)respectively . The application of multiplex PCR on some Staph.aureus isolates (8) was effectively in detection of Coa. SpA, ,TST,HIg, CLF, nucgenes by amplification at a single amplicon at (630bp, 226 bp,326bp, 937bp, 638bp, 395bp respectively).