Background: Staphylococcus aureus (S. aureus) is major cause of nosocomial infections. The accessory gene regulator (agr) system of Staphylococcus aureus controls the expression of the genes encoding extracellular virulence factors. The aim of this study was to investigate the frequency of agr genes and drug resistance in Staphylococcus aureus strains isolated from clinical samples in Tabriz hospitals. Methods: This cross-sectional study was done among a total of 100 strains isolated from clinical samples in Tabriz hospitals. Antibiotic susceptibility pattern of all isolates was determined by disk diffusion method. After DNA extraction, the presence of agr genes was investigated using Multiplex PCR. SPSS software was used to perform statistical tests. Results: Among 100 Staphylococcus aureus strains, 38 isolates were resistant to methicillin. They were susceptible to rifampin (89%) and vancomycin (86%) but showed resistance to penicillin (98%) and tetracycline (85%). The most prevalent gene was agrA (65%) followed by the agrC (29%) in strains. None of the isolates harbored the agrB and agrD genes. Conclusion: agrA and agrC genes play an important role in staphylococcal infections in clinical samples isolated from Tabriz hospitals. Also, the results showed high rates of multi-drug resistance in Staphylococcus aureus strains isolated from Tabriz hospitals. Therefore, it is recommended to limit the unnecessary uses of antibiotics.