Eight Borrelia burgdorferi strains, which had been isolated from Ixodes nipponensis and Apodemus agrarius captured in the Chungju area of Korea, were characterized by ospC gene sequence analysis. Nucleotide sequence similarity among the Chungju strains ranged from 83.6 to 100%. Deduced amino acid sequence similarity of the Chungju strains ranged from 75.4 to 100%. In a molecular phylogenetic analysis, the Chungju strains were separated from B. burgdorferi and B. garinii reference strains, and formed a cluster with B. afzelii reference strains. Three (KK2, KM4, and KK5), two (CJ2 and CJ21), and one (CJ3) Chungju strains formed clusters with OspC serotype 5, OspC serotype 8, and OspC serotype 7 reference strains, respectively. However, two Chungju strains (KK1 and KM10) formed a distinctive cluster that was separated from other strains of B. afzelii reference strains. These results suggest that Chungju strains are very heterogeneous in clonality.