We compared the performance characteristics of culture and the Cepheid Xpert vanA assay for routine surveillance of vancomycin-resistant enterococci (VRE) from rectal swabs in patients at high risk for VRE carriage. The Cepheid Xpert vanA assay had a limit of detection of 100 CFU/ml and correctly detected 101 well-characterized clinical VRE isolates with no cross-reactivity in 27 non-VRE and related culture isolates. The clinical sensitivity, specificity, positive predictive value, and negative predictive value of the Xpert vanA PCR assay were 100%, 96.9%, 91.3%, and 100%, respectively, when tested on 300 consecutively collected rectal swabs. This assay provides excellent predictive values for prompt identification of VRE-colonized patients in hospitals with relatively high rates of VRE carriage.