Introduction: Mupirocin, an inhibitor of bacterial isoleucyl-tRNA synthetase, has used to treat staphylococcal skin infections as well as to eliminate nasal carriage of Staphylococcus aureus. Mupirocin resistant Staphylococcus aureus strains are seen worldwide but there is no report from Iran. In the present study, by using phenotyping methods (disk diffusion and E-test) in combination with PCR assays, we investigate the presence of mupirocin resistance in Staphylococcus aureus isolated from patients in Tehran .   Materials & Methods: C onfirmation of the isolated strains in 4 university hospitals in Tehran as Staphylococcus aureus was done by routine laboratory tests (Gram's stain, catalase, DNase, slide and tube coagulase tests and mannitol fermentation) in combination with PCR method for femB gene, encoding a unique feature to Staphylococcus aureus. Presence of mupA gene, responsible for mupirocin resistance, was determined by PCR method. Disk diffusion and E-test were also used for demonstrating mupirocin resistance. The susceptibility testing of S. aureus isolates were carried out by the disk diffusion method according to the Clinical Laboratory Standards Institute guidelines. Moreover, PCR method was used for detection of mecA gene, which conferring resistance to methicillin and other penicillinase resistant penicillins.  Results : Identity of 94 strains as Staphylococcus aureus were confirmed by femA gene PCR and phenotypic methods. Six strains were considered resistant to mupirocin, 5 low-level and 1 high-level resistances to mupirocin. From these 6 strains, mupA gene were shown in 5 strains (DNA band with 456bp molecular weight), and in one strains this band was not detected. All mupirocin resistant strains were methicillin resistant by mecA gene PCR and disk diffusion test. Disk diffusion test had shown all mupirocin resistant strains were resistant to penicillin G , ampicillin, oxacillin, ciprofloxacin, tetracycline, trimethoprim - sulfamethoxazole, cefoxitin, clindamycin and erythromycin and resistance to other examined antibiotics were seen in some strains but all strains were shown susceptibility to vancomycin.  Conclusion: This study is the first report about mupirocin resistance in Staphylococcus aureus strains isolated from patients in Tehran and calls further studies for determination the incidence of this resistance. Also, a combination of PCR and phenotypic methods was recommended for a reliable identification of mupirocin resistance in Staphylococcus aureus strains.