In recent years, Borrelia burgdorferi sensu lato has been subdivided into three species, Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, and a new species restricted to Japan, Borrelia japonica, has been isolated from Ixodes ovatus. In addition, members of several new genomic groups have been found in America and in Europe, suggesting that there are additional genospecies. In order to study the diversity of B. burgdorferi sensu lato, we analyzed 54 isolates, cultured from humans and from different tick species and obtained from diverse geographic areas, including Europe, the United States, Japan, and the People's Republic of China. In order to investigate the genetic relationship between microorganisms that are transmitted by soft ticks and microorganisms that cause Lyme disease, we also included three strains of relapsing fever spirochetes. The method which we used was multilocus enzyme electrophoresis; 12 genetic loci were characterized on the basis of the electrophoretic mobilities of their products, and 50 distinct allele profiles (electrophoretic types) were distinguished. The mean genetic diversity per locus was 0.747. a cluster analysis of a matrix of genetic distances for pairs of electrophoretic types revealed 11 divisions that were separated at genetic distances greater than 0.65. Five of these divisions corresponded to B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. japonica, and the newly proposed species "Borrelia andersonii." Our results also confirmed that there are two additional genomic groups in Europe and at least one additional group in the United States. The relapsing fever spirochetes were were not clearly separated from the spirochetes associated with Lyme disease. In conclusion, we believe that the previously proposed subdivision of B. burgdorferi sensu lato into only four species should be reconsidered.