The incidence of methicillin-resistant Staphylococcus aureus (MRSA) has been increasing yearly at Peking Union Medical College Hospital (PUMCH). In order to understand the molecular evolution of MRSA at PUMCH, a total of 466 nonduplicate S. aureus isolates, including 302 MRSA and 164 methicillin-susceptible (MSSA) isolates recovered from 1994 to 2008 were characterized by staphylococcal cassette chromosome mec (SCCmec) typing, spa typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). The 302 MRSA isolates were classified into 12 spa types and 9 sequence types (STs). During the years from 1994 to 2000, the most predominant MRSA clone was ST239-MRSA-III-spa t037. Since 2000, ST239-MRSA-III-spa t030 has rapidly replaced t037 and become the major clone. Another clone, ST5-MRSA-II-spa t002 emerged in 2002 and constantly existed at a low prevalence rate. The 164 MSSA isolates were classified into 62 spa types and 40 STs. ST398 was the most common MLST type for MSSA, followed by ST59, ST7, ST15, and ST1. Several MSSA genotypes, including ST398, ST1, ST121, and ST59, were identical to well-known epidemic community-acquired MRSA (CA-MRSA) isolates. MLST eBURST analysis revealed that the ST5, ST59, and ST965 clones coexisted in both MRSA and MSSA, which suggested that these MRSA clones might have evolved from MSSA by the acquisition of SCCmec. Two pvl-positive ST59-MRSA-IV isolates were identified as CA-MRSA according to the clinical data. Overall, our data showed that the ST239-MRSA-III-spa t037 clone was replaced by the emerging ST239-MRSA-III-spa t030 clone, indicating a rapid change of MRSA at a tertiary care hospital in China over a 15-year period.