Mitis group streptococci (MGS) were classified based on the nucleotide sequences 16S rRNA gene (16S rDNA) and comprised 13 Streptococcus species. However, 16S rDNA homogeneity among MGS was too high to discriminate between clinical strains at the species level, notably between Streptococcus mitis, Streptococcus oralis, Streptococcus pneumoniae, and Streptococcus pseudopneumoniae. The purpose of this study was to discriminate between 37 strains of MGS isolated from Korean oral cavities using phylogenetic analysis of the DNA-dependant RNA polymerase beta‐subunit gene (rpoB). 16S rDNA and rpoB from clinical strains of MGS were sequenced using the dideoxy chain termination method and analyzed using MEGA version 5 software. The resulting phylogenetic data showed that the rpoB sequences could delineate clinical strains of MGS at the species level. Phylogenetic analysis of rpoB is therefore a useful approach for dentifying MGS at the species level.