Objective:The purpose of this study was to determine the prevalence of vancomycin-resistantStaphylococcus aureusisolated from clinical samples in Shiraz hospitals. Methods:From March to December 2012, 100S. aureusisolates (mainly from wound and blood) were collected from three hospitals in Shiraz, south of Iran. After identification ofStaphylococcus aureusby biochemical, microbiological and molecular methods, antibiotic susceptibility testing was performed by Kirby-Bauer disc diffusion test for 13 different antibiotics. Vancomycin-resistantStaphylococcus aureusisolates were determined by vancomycin agar screening test and PCR forvancomycin resistantgenes (vanAandvanB). Results:The lowest and highest resistance was seen for quinupristin-dalfopristin (n=1) and ampicillin (n=95), respectively. Vancomycin agar screening test showed that 37 isolates can grow on these media. Further study by PCR also detectedvanAand/orvanBgenes in all of these strains. Also, 19 isolates showed eithervanAorvanBbut were susceptible according to vancomycin agar screening test. In total,vanAandvanBresistant genes were detected in 34% and 37% of clinical isolates, respectively. Conclusion:The results showed that the frequency of vancomycin resistance genes (vanA, vanB) is very high inStaphylococcus aureusstrains isolated from patients insouthof Iran. Thus, urgent interventions are needed to keep the emergence and transmission of these isolates to a minimum.