2 (i)-ro, Osong-eup, Cheongwon-gun, Chungcheongbuk-do 363-951, Republic of Korea This study was conducted to investigate the molecular characteristics and genetic relatedness of vancomycin-resistant Enterococcus faecium (VREF) isolates obtained from humans and poultry in Korea. A total of 147 VREF isolates from humans (71 clinical isolates) and poultry (76 isolates) in Korea were compared with respect to their antibiotic susceptibilities, organization of the Tn1546 transposon element, detection of virulence genes (esp and hyl), pulsed-field gel electrophoresis (PFGE) typing and multilocus sequence typing (MLST). All of the human and poultry isolates had the vanA gene and 11.3 % (8/71) of the clinical isolates showed the VanB phenotype/vanA genotype. PCR mapping of the Tn1546 elements was different for isolates of the two groups: human isolates were classified into five transposon types, whereas all poultry isolates were identical to Tn1546 of E. faecium strain BM4147. The esp gene was detected in both human (93.0 %, 66/71) and poultry (26.3 %, 20/76) isolates, as was the hyl gene (human isolates: 80.3 %, 57/71; poultry isolates: 26.3 %, 20/76). Using MLST, the 71 human isolates could be divided into ten sequence types (STs) belonging to clonal complex (CC) 17 (except for one singleton). The 76 poultry isolates were categorized into 14 STs and 88.2 % (67/76) of the poultry isolates belonged to CC26. PFGE typing of the human isolates demonstrated diverse PFGE profiles among the strains. However, the PFGE patterns of the poultry isolates were possibly related to the strains collected from individual farms. These data suggest that epidemic clonal groups of human and poultry VREFs in Korea have evolved through different evolutionary processes.