Staphylococcus aureus is one of the most important pathogenic type to humans, and the most common species responsible for a wide range of diseases such as furuncles, various abscesses, wounds abscesses resulting from surgical operations, dermatitis, soft tissue inflammation, arthritis, bones inflammation, bronchial pneumonia, inflammation of internal parts of the heart and injuries caused by toxins such as toxic shock syndorome and staphylococcus aureus syndrome and food poisoning. The current study aimed by finding the genes responsible for the virulence factors in S. aureus isolates by using the Single and Multiplex PCR mechanism (technology). A total of 60 specimens (urine, burn swabs, wound swaabs) from different clinical cases were collected from patients (in different age groups) who admitted to several health centers in Al-Diwaniyah Teaching Hospital, Iraq, during a period extending from October 2016 to January 2017. Some virulence factors were investigated for 30 isolate only of MRSA using Single and Multiplex PCR for detection virulence factor genes which both coa gene encoding production of coagulase, clfA gene encoding for clumpting factor, spa gene encoding for protein A, fnbA gene encoding for fibronectin binding proteins, luks gene encoding prouction of Panton Valentine Leukocidin (PVL). Results 30 (100%) were possess coa, clfA, spa and fnbA genes, 13 (43.3%) were possess luks gene