The A and B clones of Borrelia burgdorferi sensu stricto, distinguished by outer surface protein C (ospC) gene sequences, are commonly associated with disseminated Lyme disease. To resolve phylogenetic relationships among isolates, we sequenced 68 isolates from Europe and North America at 1 chromosomal locus (16S-23S ribosomal RNA spacer) and 3 plasmid loci (ospC,dbpA, and BBD14). The ospC-A clone appeared to be highly prevalent on both continents, and isolates of this clone were uniform in DNA sequences, which suggests a recent trans-oceanic migration. The genetic homogeneity of ospC-A isolates was confirmed by sequences at 6 additional chromosomal housekeeping loci (gap, alr, glpA, xylB, ackA, and tgt). In contrast, the ospC-B group consists of genotypes distinct to each continent, indicating geographic isolation. We conclude that the ospC-A clone has dispersed rapidly and widely in the recent past. The spread of the ospC-A clone may have contributed, and likely continues to contribute, to the rise of Lyme disease incidence.