BACKGROUND Methicillin-resistant Staphylococcus aureus (MRSA) infections have become a leading cause of severe infections in both healthcare and community settings. Mutations in the rpoB gene cause resistance to rifampin (RIFR), a critical antibiotic for the treatment of multidrug-resistant Staphylococcus aureus. The aim of this study was to detect the molecular characteristics of RIFR MRSA and analyze the rpoB gene mutations involved in RIF resistance. METHODS A total of 49 RIFR MRSA and 38 RIFS MRSA isolates collected from seven cities in China were analyzed by multilocus sequence typing, staphylococcus chromosomal cassette mec (SCCmec) typing, spa typing, and rpoB gene mutations. RESULTS ST239-III-t030 (35/49, 71.4%), the major clone in RIFR MRSA isolates; ST45-IV-t116 (16/38, 42.1%), the major clone in RIFS MRSA isolates with rpoB mutations. RIFR MRSA isolates were resistant to erythromycin, ciprofloxacin, tetracycline, gentamicin, and clindamycin. By contrast, RIFS MRSA isolates with rpoB mutation were more susceptible to ciprofloxacin, tetracycline, and gentamicin. Forty-three (87.8%) isolates present the mutational change H481N and L466S, conferring 128-512 μg/mL RIF resistance. The four isolates with RIF MIC ≥ 1024 μg/mL had additional amino acid substitution: H481N, L466S, A473T (n=2); H481Y (n=2), associated with a high-level RIF resistance. Of 38 RIFS MRSA isolates, two mutations were observed, including H481N (n=37) and A477D (n=1). CONCLUSIONS In conclusion, the predominant RIFR MRSA clones in China were ST239-III-t030. Molecular characteristics, antibiotic-resistant profiles, and rpoB mutations between RIFR MRSA and RIFS MRSA were diverse. Antibiotics for treating patients with MRSA infections can be selected based on molecular characteristics.