We describe a phylogenetic investigation of Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, based on a DNA sequence analysis of the hbb gene, which encodes protein HBb, a member of the family of histone-like proteins. Because of their intimate contact with the DNA molecule, these proteins are believed to be fairly conserved through evolution. In this study we proved that the hbb gene is suitable for phylogenetic inference in the genus Borrelia. The hbb gene, which is 327 bp long and encodes 108 amino acids, was sequenced for 39 strains, including 37 strains of B. burgdorferi sensu lato, 1 strain of Borrelia turicatae, and 1 strain of Borrelia parkeri. Genetic variability was determined at the sequence level by computational analysis. Briefly, 81 substitutions were scored at the DNA level. Only 25 of these substitutions were responsible for amino acid substitutions at the translational level. The signature region for bacterial histone-like proteins was found in hbb. Although variable at the nucleotide level, it was highly conserved at the deduced amino acid level. A phylogenetic tree for the genus Borrelia that was generated from multiple sequence alignments was consistent with previously published data derived from DNA-DNA hybridization and multilocus enzyme electrophoresis analyses. The subdivision of B. burgdorferi sensu lato into five species (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, and "Borrelia andersonii") and at least four genomic groups (groups PotiB2, VS116, CA2, and DN127) was confirmed.