IxodespersulcatusservesasatickvectorforBorreliagariniiandBorreliaafzeliiinJapan;however,unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 fromIxodes tanuki, 6 fromIxodes turdus, and 1 fromIxodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction frag- ment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that theseBorreliastrains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates ofI. tanukiandC. rufo- canusas group Hk501 and all isolates ofI. turdusas group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme diseaseBorreliaspecies known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 fromI. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.