'Streptococcus bovis/Streptococcus equinus' is a large bacterial complex including different species frequently isolated from infections of humans (Streptococcus gallolyticus, Streptococcus infantarius) or animals (S. bovis, S. equinus, Streptococcus alactolyticus). The separation of S. bovis into three different biotypes has been partially correlated with genetic differentiation. In addition, recent advances in bacterial phylogeny have led to the inclusion of Streptococcus macedonicus and Streptococcus waius in this complex. The aim of this study was to improve physiological differentiation between species related to the complex and to clarify their respective phylogenetic positions. In this study, physiological, genetic and phylogenetic analyses of a set of 88 streptococcal strains were performed. The diversity of strains of S. bovis biotype II was analysed, and it was confirmed that they belong to different species, either S. equinus or S. infantarius. It was demonstrated that S. gallolyticus, S. bovis biotype II.2, S. macedonicus and S. waius form a single DNA cluster separated into three different subspecies. They are delineated by different biochemical traits, limited DNA-DNA relatedness and noticeable divergence in 16S rDNA sequences. According to the current definition of species, the names S. gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. pasteurianus subsp. nov. and S. gallolyticus subsp. macedonicus subsp. nov. are proposed for these three subspecies.