Staphylococcus aureus (S. aureus) is one of the most pathogenic bacteria isolated from milk and is one of the main causes of food poisoning outbreaks worldwide. This study was performed to investigate prevalence of S. aureus in raw cow's milk sold in dairy shops at Sohag Governorate, Egypt, determine some virulence factors and antimicrobial susceptibility of the isolates and to examine some Methicillin-resistant S. aureus (MRSA) isolates by PCR for presence of some antibiotic resistance and virulence genesincluding coa (coagulase), nuc (thermonuclease), spa (Staphylococcal protein A), mecA (a determinant of methicillin resistance) and tetK (a determinant of tetracycline resistance) genes. Therefore, a total of 300 samples of raw cow's milk were randomly collected from dairy shops in three different cities at Sohag Governorate, Egypt (El Balyana, Sohag and Tahta cities) during the period from November 2020 to August 2021. Bacteriological examination revealed that 67 samples were positive for S. aureus with percentage of (22.3%) and that (68.7%) and (31.3%) of the isolates exhibited β-hemolysis and α- hemolysis on blood agar, respectively. Also, it revealed that (46.3%) and (58.2%) of the isolates had Congo red binding (CRB) activity and DNase activity, respectively. Antimicrobial susceptibility testing for S. aureus isolates (n=67) against 9 different antibiotics revealed that they were sensitive to vancomycin (100%),  gentamycin (100%), amoxicillin/clavulinic acid (97%), ciprofloxacin (92.5%) and erythromycin (82.1%) while they showed highest resistance to oxacillin (70.1%) followed by tetracycline (67.2%), chloramphenicol (59.7%) and penicillin (47.8%). Furthermore, it revealed that (43.4%) of the isolates were resistant to 3 antibiotics or more and that (79.3%) of these multiple drug resistance (MDR) isolates were MRSA. Screening of 10 randomly selected MRSA isolates for presence of coa, nuc, spa, mecA and tetK genes by PCR revealed that all of them harbor coa, nuc and spa genes, while only (80%) and (70%) of them harbor mecA and tetK genes, respectively. In conclusion, our results indicated presence of a high prevalence of MDR S. aureus especially MRSA in raw cow's milk which represents a major threat to public health, therefore more restrictive hygienic measures must be applied during all stages of milk production.