The nucleotide sequences (534 to 546 bp) of the groEL gene, which encodes the 60-kDa heat shock protein GroEL, from 15 rickettsial strains were determined and compared. In the phylogenetic tree created by the unweighted pair group method with arithmetic averages and the neighbor-joining method, rickettsial strains could be distinguished from Ehrlichia strains. Five spotted fever group strains, four typhus group strains, and six scrub typhus group (STG) strains were differentiated as distinct entities. Unlike gltA and ompA gene analyses, differentiation between members of the genus Rickettsia and the STG rickettsiae by groEL gene analysis was possible. In comparison with 16S rRNA gene analysis, the groEL gene has a higher degree of divergence among the rickettsiae. We therefore successfully developed rapid differentiation methods, PCR-restriction fragment length polymorphism analysis and a species-specific PCR, based on the groEL gene sequences. Four Korean isolates were identified by these methods and groEL gene analysis. The results suggest that the groEL gene is useful for the identification and characterization of rickettsiae.