Methicillin resistant Staphylococcus aureus (MRSA) is one of the most important etiology agent of community and hospital acquired infections. In our country, the prevalence of MRSA is estimated to be 50%. The aim of present study was to determine the prevalence of MRSA and Vancomycin resistant Staphylococcus aureus isolates in an Iranian 1000-bed tertiary care hospital. During a period of September 2013 and June of 2014 a total of 220 nonduplicate S.aureus strains were isolated from clinical specimens. PCR and Cefoxitin disk diffusion methods were used for detection of MRSA. Vancomycin screen agar (BHI agar containing 6μ/ml Vancomycin) and E-test MIC method were used for detection of VISA VRSA. The susceptibility testing of isolates to other antibiotics was used by the disk diffusion method as recommended by CLSI. In our study 145 (65.90%) out of 220 isolates were isolated from urine and the others were isolated from other specimens such as wound and tracheal aspirates. By PCR method 105(47.72%) isolates were found to be MRSA. In our study the results of Cefoxitin disk diffusion method was in accordance to PCR. No VISA and VRSA isolates was determined by Vancomycin screenagar and E-test. The sensitivity and specificity of Cefxoitin disk diffusion method for detection of MRSA was 100%.It is concluded that the rate of MRSA and VRSA in our study was 47.72% and 0% respectively. In laboratories with limited sources for performance PCR, other methods such as Cefoxitin disk diffusion and Vancomycin screen agar are the best alternative methods for detection of MRSA, VISA and VRSA.