The genes coding for outer surface protein OspC from 22 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis were cloned and sequenced. For reference purposes, the 16S rRNA genes from 17 of these strains were sequenced after being cloned. The deduced OspC amino acid sequences were aligned with 12 published OspC sequences and revealed the presence of 48 conserved amino acids. On the basis of the alignment, OspC could be divided into an amino-terminal relatively conserved region and a relatively variable region in the central portion. The distance tree obtained divided the ospC sequences into three groups. The first group contained ospC alleles from all (n = 13) sensu stricto strains, the second group contained ospC alleles from seven Borrelia afzelii strains, and the third group contained ospC alleles from five B. afzelii and all (n = 9) Borrelia garinii strains. The ratio of the mean number of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site calculated for B. burgdorferi sensu stricto, B. garinii, and B. afzelii ospC alleles suggested that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the relatively variable region. On the basis of the comparison of 16S rRNA gene sequences, Borrelia hermsii is more closely related to B. afzelii than to B. burgdorferi sensu stricto and B. garinii. In contrast, the phylogenetic tree obtained for the B. hermsii variable major protein, Vmp33, and 18 OspC amino acid sequences suggested that Vmp33 and OspC from B. burgdorferi sensu stricto strains share a common evolutionary origin.