We sequenced homologous chromosomal loci from several North American and European isolates of the Lyme disease spirochete Borrelia burgdorferi, as well as from the relapsing fever spirochete Borrelia hermsii. Inter- and intraspecies sequence comparisons permitted the design of B. burgdorferi-specific polymerase chain reaction primers that detected all strains tested (n = 31) from diverse geographical and biological origins. Polymerase chain reaction "typing" with other unique sets of primers subdivided B. burgdorferi isolates into two groups: all North American isolates and a few European isolates made up one group, while the majority of the European and Asian isolates made up the second group. This classification may have a clinical correlate reflected in differences between "typical" Lyme borreliosis in North America and Europe.