Staphylococcus aureus has emerged as a significant public health problem as it is often responsible for intramammary infection in bovine. The emergence of MRSA in animals was from an outbreak of mastitis in cattle which represent a great economic in milk industry. The present study was carried out to genotypically characterized S. aureus isolated from subclinical bovine mastitis in different farms in EL-Sharkia Governorate. A total of 15 S. aureus isolates were obtained from 100 subclinical mastitic milk samples and subjected to PCR for detection of some virulence and antimicrobial resistance genes using oligonucleotide primers that amplified genes encoding enterotoxin genes A to E (sea, seb, sec, sed, see), coagulase gene (coa), the IgG binding region of protein A (spa) and resistance gene as factor essential for expression of methicillin resistance (femA) which was used as an internal positive control and intrinsic methicillin resistance gene (mecA). PCR amplification revealed that all S. aureus isolates were enterotoxogenic and MRSA, harbored the genes encoding staphylococcal coagulase and the genes encoding the immunoglobulin G binding region of protein A. The data in the study provided an overview on the distribution of virulence determinants of MRSA strains which contributed to bovine mastitis problem in the Egypt farm.