Resistance to mupirocin in methicillin-resistant Staphylococcus aureus (MRSA) have increased with wide use of mupirocin in many countries, but the prevalence in Korea is not well-known. The aim of this study was to determine the prevalence, antimicrobial susceptibility, and clonality of mupirocin-resistant (MUP-R) isolates from three Korean hospitals. A total of 175 MRSA isolates were collected from three university hospitals in 2009-2010. Antimicrobial susceptibility was tested by the disk diffusion and the agar dilution methods. femA, mecA and mupA genes were detected by polymerase chain reactions. Pulsed-filed gel electrophoresis (PFGE) pattern of genomic DNA was determined after digestion with SmaI. Overall, 12 among the 175 MRSA isolates were resistant to mupirocin, with prevalence ranging from 0 to 10% depending on hospitals. Three high-level (HL) and nine low-level (LL) MUR-R isolates were obtained from two hospitals. All MUP-R isolates were susceptible to rifampin and vancomycin, but were resistant to ciprofloxacin, clindamycin, and erythromycin. Eight LL and one HL MUP-R isolates were also resistant to fusidic acid. PFGE analysis showed three HL MUP-R isolates belonged to arbitrary cluster 3, 5 and 6 with 60~90% similarity compared to LL MUP-R isolates. In conclusion, the HL resistance to mupirocin was detected in two hospitals, but HL MUP-R isolates were clonally not related.