Genotypic variation and stability of isolates of vancomycin-resistant enterococci (VRE) were studied to determine genetic diversity and whether strain definition based on pulsed-field gel electrophoresis (PFGE) is applicable to an endemic setting. Twenty-two PFGE types were identified among 455 VRE isolates. One-on-one comparisons of 10 vanA Enterococcus faecium strain types all yielded > 10 band differences. Variations among vanA and vanB E. faecium isolates from individual long-term-colonized (4-160 days) patients yielded < 3 band differences for > 85% of comparisons. Comparison of all strains without grouping by vancomycin resistance types yielded two peaks of band differences: one with < 3 and one with > 10 band differences. These data show that VRE isolates were genetically closely related or very different; demonstrate that within individual patients, VRE isolates show little genetic variation; and provide empirical evidence that PFGE can be used to study the epidemiology of VRE endemicity.