A total of 123 genetically-unrelated strains of Enterococcus spp. strains (51 Enterococcus faecalis, 57 Enterococcus faecium, 10 Enterococcus gallinarum, and 5 Enterococcus casseliflavus) were phenotypically identified by biochemical profiles and by using an automated method. The strains were also analyzed by a PCR assay to assess the accuracy of the phenotypically-based methods for identification of Enterococcus spp. With this aim, a PCR assay using different cell targets, which allows simultaneous detection of glycopeptide-resistant genotypes as well as identification to the species level by means of different gene targets, was used as the gold standard method. All 51 strains of E. faecalis were correctly identified, whereas 48 of 57 strains (84.2%) of E. faecium, were correctly identified. All of the strains of E. gallinarum and 3 out of 5 strains of E. casseliflavus were also correctly identified. The overall results showed that it is possible to identify Enterococcus spp. at the molecular level in less than 30 hours, compared with the 48-96 hours required for the phenotypically-based methods. The excellent accuracy of the PCR assay in identifying these species, particularly E. faecium, must also be emphasized. These findings may have implications for the routine clinical identification of enterococci species.