Glycopeptide-resistant enterococci (GRE) are important causes of nosocomial infections in the United States and in Europe. Rapid detection of GRE is essential for the implementation of appropriate control measures to prevent the spread of GRE. In this study, we compared the reliability of 3 different methods, VITEK 2 automated system (bioMérieux), a conventional multiplex polymerase chain reaction (m-PCR) protocol, and a real-time PCR protocol performed on the LightCycler system (Roche) for identification of GRE in the routine microbiology laboratory. Species identification and glycopeptide resistance determination was tested with 80 enterococcal isolates with different glycopeptide resistance phenotypes. With the VITEK 2 system, 39% of the strains were correctly identified to species level. Resistance to vancomycin was detected in all isolates; however, discrepancies occurred in the correct detection of teicoplanin resistance. The PCR protocols proved to be suitable for detecting clinically relevant GRE; 90% of the isolates studied were correctly identified with the conventional m-PCR and 100% of vanA and vanB isolates with the real-time PCR protocol, respectively. High specificity and rapidity make the real-time PCR assays superior tools for identification of GRE in clinical samples; however, they do not have the ability to detect vanC.