Aims: To compare different tests in the identification of Enterococcus durans , E. hirae and E. villorum strains. These bacteria belong to the E. faecium species group and are phylogenetically closely related, as evidenced by 16S rRNA sequence homologies of over 98.8%. Methods and Results: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of whole-cell protein, tRNA interpacer polymerase chain reaction (PCR) and arbitrarily-primed (D11344-primed AP) -PCR analysis correctly identified all three species in a collection of strains from very diverse origins. In contrast, biochemical reactions only allowed the unequivocal differentiation of the three species as a group from the other enterococci. Within this group, D-xylose acidification can be used to differentiate E. villorum , but exceptions occur. Strains highly susceptible to clindamycin can be identified as E. durans , but many strains of this species cannot be differentiated from E. hirae and E. villorum due to acquired resistance. Conclusions: Despite their close relationship, E. durans , E. hirae and E. villorum can be differentiated by genomic methods and by whole-cell protein analysis. Significance and Impact of the Study: Only a minority of strains of these three enterococcal species can be identified reliably by the currently available and commonly applied phenotypic tests.