Sequence type (ST) 239 with SCCmec type III methicillin-resistant Staphylococcus aureus (ST239-MRSA-III) is the most predominant multidrug-resistant clone in China. The subclone ST239-MRSA-III-t037 has been gradually replaced with ST239-MRSA-III-t030 since 2000. Subclones are characterized by drug resistance profiles. However, the mechanisms of the clonal dynamics and determinants of distinct drug resistance remain poorly understood. In the present study, 12 ST239-MRSA-III-t030 and 12 ST239-MRSA-III-t037 strains were collected from Chongqing, Guangzhou, and Shanghai; these strains were selected and investigated in terms of t030/t037 strain pairs. Independent growth curve assay revealed that the ST239-MRSA-III-t030 strains grew more rapidly, with significantly shorter doubling times, than the ST239-MRSA-III-t037 strains (p < 0.001). The ST239-MRSA-III-t037 strains exhibited slightly to moderately higher (3-13%) fitness cost than the ST239-MRSA-III-t030 strains in a competition assay in vitro. The ST239-MRSA-III-t037 strains yielded lower bacterial loads in the kidneys of the infected mice than the ST239-MRSA-III-t030 rivals in a coinfection assay (p < 0.05). The ST239-MRSA-III-t030 strains were resistant to rifampicin but susceptible to trimethoprim/sulfamethoxazole (SXT). In contrast, the ST239-MRSA-III-t037 strains were susceptible to rifampicin but resistant to SXT. The genetic determinants of the resistance to rifampicin and SXT in the MRSA strains were determined. Our results suggest that the relatively low fitness cost and characteristic drug resistance phenotype can help explain the current predominance of these ST239-MRSA-III-t030 strains in Chinese hospitals.