To enhance the diagnosis of Staphylococcus aureus mastitis and its prospective antibiotic resistance in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for concurrent species identifycation, detection antibiotic resistant phenotypically and genotypically to penicillin (blaZ gene), gentamycin (aac(6') aph (2'')genes), and tetracycline (tetK, gene) and recognize the toxogenicpatterns of  S. aureus in cow's milk with recurrent clinical mastitis. Bacterial culturing was carried out on 179 bovine recurrent clinical mastitic milk samples resulted in 84 (46.9%) coagulase positive staph isolates, while 69 (38.5%) confirmed by molecular identification by PCR as S. aureus. Also some virulence factors as enterotoxin A and B (Sea and Seb) genes were determined in 7(10.1%) and 1 (1.4%) respectively in confirmed isolates. As well as determined drugs resistance and their genes. The results of antimicrobial susceptibility tests showed 100%. 43.5% and 58% resistance for penicillin, gentamycin and tetracycline respectively. While genotypically were 100%, 40.6% and 53.6% for (blaZ, aac (6') aph (2'') and tetK, respectively. This study reports the presence of multidrug resistant S. aureusin recurrent clinical mastitis with highly virulent toxic genes that could be a major obstacle in the treatment and control of mastitis in dairy farms causing highly economic impacts and recommended that the polymerase chain reaction (PCR)-based assays to detect pathogens associated with mastitis and that it has several advantages, including rapid results and high sensitivity.