Mutations in mutS and mutL, which encode DNA mismatch repair (MMR) proteins, can confer hypermutator phenotypes and may facilitate the emergence of mutational antibiotic resistance in bacteria. Linezolid-resistant enterococci (LRE) rarely emerge during therapy and contain mutations in 23S rRNA genes. As enterococci with defective MMR could be prone to the development of oxazolidinone resistance mutations, we investigated 13 clinical isolates of Enterococcus faecium, including 2 LRE, for mutations in mutSL. A 4,944-bp fragment spanning mutSL was sequenced from two pairs of linezolid-resistant (MICs, 64 μg/ml) and linezolid-susceptible (MICs, 2 μg/ml) E. faecium isolates (one pair from Austria and one pair from the United Kingdom) identical by pulsed-field gel electrophoresis. The pairs represented distinct strains in which linezolid resistance had emerged during therapy. The MutSL peptides of all four isolates had amino acid substitutions compared with the sequence of E. faecium strain DO (used for genome sequencing). These were Val352Ile (one pair of isolates only) and Met628Leu in MutS and Leu387Pro, Tyr406Phe, Thr415Ser, Phe427Leu, and Phe565Ile in MutL. The significance of these changes remains unknown; these isolates did not show a demonstrable hypermutator phenotype. The same substitutions were found in two of nine geographically diverse linezolid-susceptible enterococcal isolates; the other seven isolates had MutSL sequences identical to that of strain DO. Multilocus sequence typing revealed that all isolates with alternate MutSL peptides belonged to a distinct lineage of a prevalent E. faecium clonal complex, designated CC17. Further studies are needed to investigate the prevalence of these MutSL mutations and their possible roles in the emergence of E. faecium strains resistant to oxazolidinones and other antibiotic classes.