ABSTRACT Vancomycin-resistant Enterococcus faecium (VRE) has become an important health care-associated pathogen because of its rapid spread, limited therapeutic options, and possible transfer of vancomycin resistance to more-virulent pathogens. In this study, we compared the ability to detect clonal relationships among VRE isolates by an automated repetitive-sequence-based PCR (Rep-PCR) system (DiversiLab system) to pulsed-field gel electrophoresis (PFGE), the reference method for molecular typing of VRE. Two sets of VRE isolates evaluated in this study were collected by active microbial surveillance at a large teaching hospital in Taiwan during 2008. The first set included 90 isolates randomly selected from the surveillance cohort. The first set consisted of 34 pulsotypes and 10 Rep-PCR types. There was good correlation between the two methods ( P < 0.001). The second set included 68 VRE isolates collected from eight clusters of colonization. A dominant clone was detected in five out of eight clusters by both methods. Two clusters were characterized by Rep-PCR as being caused by a dominant clone, whereas PFGE showed polyclonal origins. One cluster was shown to be polyclonal by both methods. A single Rep-PCR clone type was detected among 12 of 14 vancomycin-intermediate enterococci, whereas PFGE detected six pulsotypes. In conclusion, the Rep-PCR method correlated well with PFGE typing but was less discriminative than PFGE in defining clonal relationships. The ease of use and more rapid turnaround time of Rep-PCR compared to PFGE offers a rapid screening method to detect outbreaks of VRE and more rapidly implement control measures. PFGE remains the preferred method to confirm clonal spread.