Effective management of hospital-acquired infections caused by necessitates a comprehensive understanding of bacterial characteristics. The genotyping of clinical samples of methicillin-resistant (MRSA) isolates plays a crucial role in understanding the pathogen's epidemiology, etiology, and antibiotic resistance patterns. This study investigated the genotyping and antibiotic resistance profiles of clinically isolated strains from different hospitals in Isfahan, Iran. Sixty-three MRSA isolates were analyzed using the disc diffusion method. After DNA extraction, multilocus sequence typing (MLST) was performed using seven housekeeping genes, revealing genetic diversity. Six isolates were selected based on their resistance patterns for MLST. The most frequent isolates were detected from wounds (41.3%), and the lowest frequency was from synovial samples (1.6%). Based on the antibiotic resistance pattern, the highest antibiotic resistance of isolates was related to tetracycline, ciprofloxacin, and clindamycin at 68.3%, 44.4%, and 44.4%, respectively. In contrast, 96.8% and 95.2% of the isolates were sensitive to nitrofurantoin and linezolid. Among resistant isolates, six sequence types (STs) were identified, including ST74, ST239, ST805, ST531, ST859, and ST5. This study highlights the prevalence, antibiotic resistance, and genetic diversity of MRSA isolates in Isfahan, Iran. The identification of clonal complexes (e.g., CC5, CC8, CC30) suggests clonal spread, emphasizing the importance of surveillance and prevention strategies.