Nine Borrelia afzelii strains, which had been isolated from two vectors, Ixodes nipponensis and Apodemus agrarius in Chungju, Korea, were characterized by PCR‐RFLP analyses of ospC genes and rrf (5S)‐rrl (23S) intergenic spacer. DraI restriction patterns of Chungju strains were identical to those of B. afzelii VS461. But MseI restriction patterns of rrf(5S)‐rrl (23S) intergenic spacer genes of KK2, KM4, KK5 differed from those of previously reported B. burgdorferi sensu lato strains. Nine Chungju strains were classified with four distinct ospC RFLP patterns, which differed from the eight ospC RFLP patterns (A‐1 to A‐8) of previously reported B. afzelii. Moreover, five additional restriction patterns were deduced from published ospC sequences of reference strains. These results suggest that Chungju strains are very heterogeneous.