ABSTRACTComparative analysis ofospCgenes from 127Borrelia burgdorferisensu stricto strains collected in European and North American regions where Lyme disease is endemic and where it is not endemic revealed a close relatedness of geographically distinct populations.ospCalleles A, B, and L were detected on both continents in vectors and hosts, including humans. SixospCalleles, A, B, L, Q, R, and V, were prevalent in Europe; 4 of them were detected in samples of human origin. TenospCalleles, A, B, D, E3, F, G, H, H3, I3, and M, were identified in the far-western United States. FourospCalleles, B, G, H, and L, were abundant in the southeastern United States. Here we present the first expanded analysis ofospCalleles ofB. burgdorferistrains from the southeastern United States with respect to their relatedness to strains from other North American and European localities. We demonstrate thatospCgenotypes commonly associated with human Lyme disease in European and North American regions where the disease is endemic were detected inB. burgdorferistrains isolated from the non-human-biting tickIxodes affinisand rodent hosts in the southeastern United States. We discovered that someospCalleles previously known only from Europe are widely distributed in the southeastern United States, a finding that confirms the hypothesis of transoceanic migration ofBorreliaspecies.