The usefulness and reliability of partial sequence analysis of the manganese-dependent superoxide dismutase gene (sodA), autolysin (lytA) gene amplification and species-specific PCR based on the D-alanine:D-alanine ligase (ddl) gene for differentiating each member of the mitis group of the genus Streptococcus was investigated. On the phylogenetic tree based on sodA partial sequences (366 bp) from 96 strains, including all species currently within the mitis group isolated in different geographic areas (mainly Japan and the UK), eight well separated clusters were generated corresponding to recognized species, and all strains fell into those clusters to which they had also been assigned by DNA-DNA hybridization. The Streptococcus pneumoniae sub-cluster was located within the Streptococcus mitis cluster, but the sodA gene of S. pneumoniae was very conserved and therefore could be separated from all other species examined. Furthermore, the lytA gene amplification approach could also be used to differentiate S. pneumoniae from other species. The species-specific amplification product of the ddl gene was successfully detected in Streptococcus sanguinis and Streptococcus gordonii, but failed to be detected in some strains of Streptococcus oralis including the type strain and S. mitis. We conclude that the partial sequence analysis of the sodA gene could be applied globally as a reliable and easy method for the accurate identification of all species currently within the mitis group.