Subclinical mastitis is considered one of the most costly diseases in dairy production throughout the world; therefore, more attention has been focused on diagnosis of subclinical mastitis. This study was conducted to establish the prevalence of subclinical mastitis (SCM) in two dairy herds located in Ismailia and El-Sharakia Governorates with isolation of staphylococci as a causative agent of subclinical mastitis. About 230 quarter milk samples (QMS) were collected from apparently healthy hundred cows and subjected to California Mastitis Test (CMT), the positive samples were examined by bacterial culture and PCR to identify staphylococcus aureus as a causative agent of subclinical mastitis. The results howed that 43.5 % of the examined QMS had subclinical mastitis, all positive CMT samples were contaminated with staphylococci, biochemical identification of staphylococcal isolates revealed that 68.05 % and 21.3 % of the examined isolates were S. aureus and coagulase negative staphylococci(CNS), respectively. By using PCR technique, gene encoded for S. aureus was confirmed in 4.35 % of the examined isolates