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Background Methicillin-resistant Staphylococcus aureus (MRSA) strains are known as one of the most important multidrug-resistant organisms causing infections in humans and animals. The objectives of this experimental study were to characterize the clonality and antibiotic resistance of MRSA strains isolated from patients in 2 different cities in Iran. Methods During 2 years, a total of 536 S. aureus isolates were collected from 2 reference hospitals in Tehran and Isfahan and were identified as MRSA using specific primers. The antibiotic susceptibility and their clonality were determined using the PhenePlate typing system. Furthermore, the presence of different classes of prophages and the structure of staphylococcal cassette chromosome mec elements and cassette chromosome recombinases types were characterized. Results Of the 536 strains, 129 MRSA were identified using species-specific primers and discriminated into 26 PhenePlate types consisted of 12 common types (CTs) and 14 single types, in which CT2 was the predominant type and 6 CTs were common among MRSA isolated in both cities. Staphylococcal cassette chromosome mec types III and IV were also detected in 89% and 11% of the strains, and SGF prophage type was the dominant one. Thirty-four antibiotic patterns were detected among the MRSA strains, and none of the isolates showed resistance to linezolid, quinupristin-dalfopristin, and vancomycin. Conclusions High prevalence of antibiotic-resistant common clonal groups of MRSA strains in 2 different cities in this study indicated the spread of these clonal types in north and center of Iran and highlighted the common origin of such strains, which are believed to be endemic in various sources.