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Haenam strains of Borrelia burgdorferi, which had been isolated from Ixodes granulatus and Apodemus agrarius in Haenam, Korea, were characterized by PCR-RFLP analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons and by sequence analysis of the 16S rRNA gene (rDNA). The Msel and Dral restriction patterns of the 5S-23S intergenic spacer amplicons of Haenam strains differed from those of other B. burgdorferi sensu lato strains. Furthermore, in the phylogenetic tree based on the 16S rDNA sequences, Haenam strains formed a distinctive cluster, clearly separated from the other members of B. burgdorferi sensu lato. These results suggest that, apart from Borrelia garinii and Borrelia afzelii, other genotypes of B. burgdorferi sensu lato exist in Korea and the Haenam strain is a newly identified one.