Different strains related to Vibrio splendidus have been associated with infection of aquatic animals. An epidemiological study of V splendidus strains associated with Crassostrea gigas mortalities demonstrated genetic diversity within this group and suggested its polyphyletic nature. Recently 4 species, V lentus, V chagasii, V. pomeroyi and V kanaloae, phenotypically related to V splendidus, have been described, although biochemical methods do not clearly discriminate species within this group. Here, we propose a polyphasic approach to investigate their taxonomic relationships. Phylogenetic analysis of V splendidus-related strains was carried out using the nucleotide sequences of 16S ribosomal DNA (16S rDNA) and gyrase B subunit (gyrB) genes. Species delineation based on 16S rDNA-sequencing is limited because of divergence between cistrons, roughly equivalent to divergence between strains. Despite a high level of sequence similarity, strains were separated into 2 clades. In the phylogenetic tree constructed on the basis of gyrB gene sequences, strains were separated into 5 independent clusters containing V splendidus, V lentus, V chagasii-type strains and a putative new genomic species. This phylogenetic grouping was almost congruent with that based on DNA-DNA hybridisation analysis. V pomeroyi, V kanaloae and V tasmaniensis-type strains clustered together in a fifth clade. The gyrB gene-sequencing approach is discussed as an alternative for investigating the taxonomy of Vibrio species.