Mastitis is a multifactorial and ubiquitous disease that results from interactions between the host, environment, and infectious agents leading to extensive economic losses. The objectives of this study were to estimate the prevalence of S. aureus in clinical and subclinical mastitis in Holstein dairy cows and determine the susceptibility of S. aureus isolatesagainst different antibiotics for screening of antibiotic resistance genes. A total of 415 Holstein dairy cows were randomly selected from three Egyptian governorates. Selected cows were examined for clinical and subclinical mastitis during the period from October 2014 to June 2018. Milk samples were examined for the presence of S. aureus. The S. aureus isolates were subjected to antibiotic sensitivity, molecular identification, and detection of the antibiotic resistance gene.The prevalence of S. aureus isolated from milk samples was 15.4% of which, 14.3% in clinical cases, and 15.7% in subclinical cases.The antibiogram of S. aureus isolates against 13 antibiotics using the disc-diffusion method revealed the highest rate of resistance to Oxacillin (OX) (96.7%), followed by Ampicillin (AM),Cefoxitin (FOX) (93.3%, each), Tetracycline (TE) (73.3%), Cefotaxime (CTX) (70%), Ampicillin/Sulbactam (SAM) (66.7%), Erythromycin (E) and Sulphamethoxazole/Trimethoprim (SXT) (56.7%, each), Gentamicin (GM) (53.3%), Ofloxacin (OFX) (40%), Chloramphenicol (C) (36.7%), Ciprofloxacin (CIP) (30%) and finally Vancomycin (VA) (0%). Molecular PCR assay revealed that all the 16 S. aureus isolates (100%) carried mecA gene, while 15 out of 16 isolates (93.7%) carried blaZ genebut, 8 out of 16 (50%) carried tetK gene, and only one isolate (0.06%) carried fexA gene. Uncontrolled uses of antibiotics in the treatment of mastitis should be restricted and increase awareness about the risk of antimicrobial-resistant bacteria in milk.