For many years Enterococcus faecium was considered a commensal of the digestive tract, which only sporadically caused opportunistic infections in severely ill patients. Over the last two decades, vancomycin resistant E. faecium (VREF) has emerged worldwide as an important cause of nosocomial infections, especially in immunocompromised patients. The global VRE epidemic was preceded by the emergence of ampicillin resistant E. faecium (AREfm) in the United States in the early 1980s. Molecular epidemiological studies of both human- and animal-derived E. faecium isolates using multilocus sequence typing (MLST) revealed the existence of host specific genogroups, including a specific genetic lineage associated with hospital related isolates. These strains were characterized by ampicillin and quinolone resistance. In addition, the presence of the variant esp gene was strongly associated with these outbreak strains. In E. faecalis, esp is contained on a so-called pathogenicity island (PAI) and is considered a putative virulence factor. This thesis describes the development of a rapid and cheap identification scheme for enterococci, which is a prerequisite to take appropriate infection control measures. Furthermore, a rapid molecular typing scheme designated multiple locus variable number tandem repeat analysis (MLVA) has been developed to determine genetic relatedness of E. faecium isolates. Clustering of MLVA profiles confirmed the host-specific genogroups found by MLST, including the hospital specific genetic lineage. Comparison of MLVA with Pulsed Field Gel Electrophoresis (PFGE), considered the “gold standard” typing method for infection control programs in hospitals, revealed that both methods were highly concordant in assigning genotypes and comparable in costs. PFGE revealed a higher discriminatory power, but MLVA was faster. Analysis of the up- and downstream regions of the esp gene among several isolates demonstrated that, like in E. faecalis, esp of E. faecium is contained on a putative pathogenicity island (PAI) with remarkable sequence heterogeneity including insertions, deletions and mutations. The E. faecium PAI has, so far, only been detected in outbreak related and clinical isolates, suggesting a role in nosocomial epidemicity. The population structure of E. faecium was elucidated with MLST and the existence of a distinct clonal complex, designated clonal complex-17 (CC17), associated with the majority of hospital outbreaks and clinical infections in five continents, was documented. This complex is correlated with ampicillin and quinolone resistance and the presence of the PAI. A retrospective study in the UMCU revealed ecological replacement of E. faecalis by CC17 E. faecium, illustrated by a decrease in total numbers of invasive enterococcal infections but an increase in proportions of invasive AREfm. Furthermore, the ratio E. faecium/E. faecalis among bloodstream isolates changed in favor of E. faecium. A similar increase of invasive AREfm was observed in hospitals dispersed over the Netherlands, although this increase was more pronounced in university hospitals than in non-university hospitals. MLVA typing of bloodstream isolates revealed clonal spread of 4 MLVA types, which seems associated with acquisition of the esp gene in two genotypes.