BACKGROUND: For the early diagnosis of tsutsugamushi disease, polymerase chain reaction (PCR) using skin specimen might be a precise and useful diagnostic tool. The purpose of this study is to detect and identify the serotype of the Orientia tsutsugamushi from the skin lesions of the patients with tsutsugamushi disease by nested PCR. METHODS: Nested PCR was used to diagnose tsutsugamushi disease and identify the serotype of the O. tsutsugamushi; Karp, Kato, Gilliam or Boryong/Kuroki. The primer sets were derived from serotype-specific DNA sequences encoding the 56kDa antigen of O. tsutsugamushi. The PCR products were analyzed by using direct cyclic sequencing. RESULTS: The serotype-specific DNA bands with 1% agarose gel electrophoresis of amplified DNAs by nested PCR were observed in all 11 skin biopsy specimens from 8 patients with tsutsugamushi disease. Among 8 patients, 7 were proved to be infected with Boryong/Kuroki strains and one with Karp. One Karp strain showed one base mutation with amino acid sequence variation, but all the Boryong/ Kuroki strains showed no mutation. CONCLUSION: We suggest that serotype-specific nested PCR is a simple, rapid and precise diagnostic method, and useful for early diagnosis of tsutsugamushi disease. Furthermore, we might detect the sequence variations of serotype-specific DNA sequences encoding 56-kDa antigen among strains.