This study compared the molecular characteristics of vancomycin-resistant Enterococcus faecium (VREF) isolates recovered from 20 non-tertiary-care hospitals (36 isolates) and three tertiary-care hospitals (26 isolates) in diverse geographical areas of Korea from October 2010 to April 2011. All isolates carried the vanA gene only, but 42% and 73% of non-tertiary and tertiary-care isolates expressed the VanB phenotype (teicoplanin minimum inhibitory concentration ⩽16 μg/ml). All isolates harboured insertion sequences, IS1542 and IS1216V, within Tn1546. The isolates from tertiary-care hospitals tended to have reduced Tn1546 lengths by deletion of sequences adjacent to IS elements. Multilocus sequence typing revealed eight sequence types within clonal complex 17 (CC17), but DNA fingerprinting by rep-PCR did not show clonal relatedness between the intra- and inter-hospital isolates. These results suggest that vanA, which has prevailed in tertiary-care hospitals of Korea since the 1990s, had been transferred horizontally to non-tertiary-care hospitals while the genetic rearrangement driven by evolutionary adaptation to adverse environments may have occurred in tertiary-care hospitals.