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We determined the nucleotide sequences (329 bp) of the rpoB DNAs from 22 reference strains of Borrelia. No insertions or deletions were observed. Deduced amino acid sequences of amplified rpoB DNA comprised 109 amino acid residues (N(450) to M(558) [Escherichia coli numbering]). All amino acid sequences were identical with the exception of those of Borrelia lusitaniae PotiB2 (T(461)–>A) and B. bissettii DN127 (I(498)–>V). Each species of B. burgdorferi sensu lato was differentiated as a distinct entity in the phylogenetic tree constructed by a neighbor-joining method. B. burgdorferi sensu lato could be distinguished from B. turicatae and B. hermsii, which are associated with relapsing fever. Seventeen Korean isolates could be identified by PCR-linked direct sequencing and restriction analysis of the rpoB DNA. These results suggest that rpoB DNA is useful for identification and characterization of Borrelia. In addition, we developed the rapid species identification method using the species-specific primer sets based on rpoB gene sequences.