Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to investigate the genetic relatedness of a total of 41 Enterococcus faecium isolates from different backgrounds (hospital outbreaks, n = 9; documented sporadic infections, n = 10; asymptomatic sporadic carriage of hospitalized patients, n = 9; healthy persons, n = 3; non-human sources, n = 10) over the period 1996-2004 in comparison with clones that have spread in Italy since 1993. Thirty six isolates were vancomycin-resistant and five were vancomycin-susceptible. eBURST analysis of MLST sequence types generated two groups: (1) group 1 (27 isolates) forming a clonal complex (CC17) with the predicted founder corresponding to ST17, a genotype identified in 1994, that included esp-positive and -negative clones isolated from hospitalized patients; and (2) group 2 (14 isolates) including esp-negative clones from different sources (hospitalized patients, healthy persons and non-human sources). The hyl gene was found in five strains with different PFGE types, all belonging to group 1, whereas cylA, gelE, and asa1, were not detected in any of the isolates. Our data showed that the evolution of the MLST C1 epidemic lineage has been continuing in several Italian areas and generating new clones with different PFGE patterns. The main, though not the sole, mechanism that has driven this evolution was confirmed to be linked to the presence of the esp gene.