The organization of the ribosomal genes is unique in Borrelia burgdorferi in that the rrl (23S) and rrf (5S) genes are tandemly duplicated. We took advantage of this uniqueness to assess the restriction polymorphism of PCR products obtained with primers at the 3' end of the first rrf gene and at the 5' end of the second rrl gene. An amplicon that was 226 to 266 bp long was generated from 99 to 100 B. burgdorferi sensu lato strains. The nuclease MseI restriction polymorphism of the amplicons provided a useful tool for identifying B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii (formerly group VS461), and Borrelia japonica (formerly group F63B). Furthermore, it allowed us to recognize four new genomic groups, which were confirmed by DNA-DNA hybridization data. Two of these genomic groups comprised European strains, and the other two groups contained American strains. The American genomic groups involved vectors with enzootic cycles quite different from those of B. burgdorferi sensu stricto, which previously was the only Lyme disease Borrelia species known to occur in the United States. Our method could be used for rapid screening of strain collections and for epidemiological and medical purposes.