Background: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most pathogens associated with health care. Molecular typing methods are vital for outbreak investigations of MRSA. The aim of this study was characterization of SCCmec, spa types and multidrug resistant of methicillin-resistant Staphylococcus aureus isolates in Ahvaz, Iran. Methods: A total of 50 MRSA isolates were determined by using the phenotypic method and mecA gene. Antibiotic resistance profile and SCCmec types were screened using disc diffusion method and PCR, respectively. For spa typing of MRSA isolates, two molecular typing methods including the PCR-sequencing and high-resolution melting (HRM) analysis were used. Results: In the present study, the highest sensitivity of MRSA was to vancomycin and linezolid and the lowest to clindamycin. In the MRSA isolates, 22% were XDR and 78% were MDR. SCCmec type III was found commonly among MRSA. Based on PCR-sequencing and HRM results, 10 different spa types were identified. The spa types t037 and t030 were the most common in this study. Conclusion: This study emphasizes the spa variation among MRSA isolates, which may be considered as an important criterion when treating staphylococcal infections. Accurate and early detection of MDR, XDR, or even PDR MRSA isolates strains must be commenced by all clinical microbiology laboratories to reduce the menace of antimicrobial resistance.