Genomic, taxonomic and biochemical studies were performed on two strains of α-haemolytic streptococci that showed them to be clustered with major members of the Streptococcus mitis group. These Gram-stain-positive strains were isolated from tooth surfaces of caries-free humans and showed the classical spherical shape of streptococcal species growing in chains. Sequence analysis from concatenated 16S and 23S rRNA gene and sodA genes showed that these strains belonged to the mitis group, but both of them clustered into a new phylogenetic branch. The genomes of these two isolates were sequenced, and whole-genome average nucleotide identity (ANI) demonstrated that these strains significantly differed from any streptococcal species, showing ANI values under 91 % even when compared with the phylogenetically closest species such as Streptococcus oralis and S. mitis. Biochemically, the two isolates also showed distinct metabolic features relative to closely related species, like α-galactosidase activity. From the results of the present study, the name Streptococcus dentisani sp. nov. is proposed to accommodate these novel strains, which have been deposited in open collections at the Spanish type Culture Collection (CECT) and Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ), being respectively identified as Streptococcus dentisani Str. 7746 ( = CECT 8313 = DSM 27089) and Streptococcus dentisani Str. 7747(T) ( = CECT 8312(T) = DSM 27088(T)).