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ABSTRACTMultilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) with SmaI were used to subtype 55 isolates of Campylobacter jejunifrom a diverse range of human and animal sources previously characterized by multilocus enzyme electrophoresis (MEE). MEE and MLST targeted 11 and 7 loci, respectively, and all loci were unique to each method. MEE, MLST, and PFGE identified 40, 37, and 48 discrete subtypes, respectively, with many of the subtypes occurring only once within the data set. Simpson's indices of diversity were calculated to be 0.979, 0.966, and 0.994 for MEE, MLST, and PFGE, respectively, demonstrating that MEE and MLST had similar discriminatory powers but that PFGE was more discriminatory. Allele diversity was higher in the MLST loci; individual single-locus diversities for the 11 MEE loci and the 7 MLST loci were 0.491 and 0.854, respectively. The clonal complexes recognized by MLST correlated with the strain associations previously recognized by MEE and contained some isolates indistinguishable by PFGE. Many clusters contained isolates from diverse geographical regions and from both humans and animals. These results demonstrate the usefulness of MLST for investigation of the global epidemiology of this important pathogen and illustrate its potential to identify indistinguishable strains or clones in geographically distinct regions.