Analysis of 16S rRNA gene sequences has been the method generally used to study the evolution and phylogeny of bacteria. Phylogenetic analysis of the 16S rRNA gene has shown the position of the genus Helicobacter in the epsilon-subclass of the Proteobacteria. Because 16S rRNA-based phylogeny does not always correspond to the results of polyphasic taxonomy, and the related species cannot always be separated, new phylogenetic markers for Helicobacter species are needed. In this study, conserved partial (600 bp) 60 kDa heat-shock protein (HSP60) sequences were used to study the phylogeny of 37 strains of gastric and enterohepatic Helicobacter species, including type strains of 15 Helicobacter species with validly published names, reference strains of flexispira taxa and Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis and canine flexispira strains. The partial HSP60 gene sequence proved to be a useful phylogenetic marker for the genus Helicobacter, providing a means of differentiating all 15 Helicobacter species analysed. In the resulting phylogenetic tree, gastric Helicobacter species and enterohepatic species with flexispira morphology formed tight, separate clusters. In general, HSP60 sequence similarities between Helicobacter species were significantly lower than the corresponding 16S rRNA gene sequence similarities, indicating a better resolution for species identification. In addition, a specific PCR method for identifying H. salomonis was developed based on the partial HSP60 sequence.