Staphylococcus aureus is one of the main significant human pathogens which can produce various toxins such as Panton-Valentine Leukocidin (PVL) which is known as a prominent toxin associated with S. aureus infections. PVL-positive strains can cause a wide variety of skin, soft tissue, necrotizing pneumonia, fasciitis and life-threatening infections. Therefore, the aim of this study was evaluating the molecular characteristics of PVL-positive strains such as the presence of mecA, SCCmec types, agr types and exfoliative toxin genes. In this study, a total of 152 S. aureus strains were collected from clinical samples of patients who referred to Isfahan’s Alzahra hospital (Iran). The isolates were confirmed phenotypically by conventional methods and then PVL-positive isolates were identified by PCR molecular test. Thereafter, antibiotic resistance pattern, agr groups (I, II, III, and IV), exfoliative toxins (eta and etb), mecA gene and SCCmec various types were carried out. Totally, 52 (34.2%) of strains were positive for PVL. Six PVL-positive strains harbored mecA gene, one strain had SCCmec I, and 5 strains SCCmec type IV. The highest ratio of agr groups belonged to group (I) and the (eta) gene was also detected in 18 isolates. The PVL-positive S. aureus strains can cause more serious infections, so identification of the genetic characteristics and antibiotic resistance monitoring of these strains is necessary.