Vancomycin Resistant Enterococci (VRE) are a major cause of nosocomial infections. The purpose of this study was the investigation of the genetic background of vancomycin resistance in VRE strains isolated from various clinical sources in a major hospital in Bucharest. Identification of Enterococcus faecium and E. faecalis at the species level was performed using multiplex PCR for species specific ddl (D-alanine-D-alanine ligase) genes, as for 64% of the strains the VITEK system revealed only the genus of the strains. We used a multiplex PCR approach, using primers targeting the vanA, vanB, vanC, vanD, vanE, vanG genes, revealing the presence of vanA and vanB genes, To the best of our knowledge, this is the first description of vanA and vanB genes in Romania. This specific and sensitive technique allows detection of glycopeptide-resistant strains, that may escape phenotypebased automated rapid methods.