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Viridans group Streptococci (VGS) are pathogens responsible for various infections such as endocarditis and septicemia. The GVS include more than 26 species grouped into five subgroups: mutans, anginosus, salivarius, sanguinis and mitis. Conventional methods are insufficient for the identification of these species. The use of molecular methods has opened new perspectives in this field. The study conducted aims to identify SGVs using helix 54 of domain III of 23S rRNA and to compare phylogenetic relationships based on these sequences with those of the 16S-23S intergenic region. For all 25 SGV strains studied, the 16S-23S intergenic region and helix 54 were amplified. Sequence analysis of the latter allowed identification of all strains studied to a species level, except those belonging to the mitis subgroup. This proves that the sequencing of helix 54 of domain III of 23S rRNA would be a reliable method for the identification, at a species level, of strains belonging to the anginosus, mutans and sanguinis subgroups.