Vibrio alginolyticus,a member of the core group of the genus Vibrio,also called V.harveyi group,shares high phenotypic and genotypic resemblance with the other species of this group.It is very difficult to identify strains of V.alginolyticus from others.The housekeeping genes and various automated systems based on biochemical characteristics of bacteria provide more alternatives for identification.In this study,we compared the resolution of 16S rRNA gene,toxR gene,pyrH gene and VITEK 2 COMPACT GN card on V.alginolyticus-related isolates.There are multiple copies of 16S rRNA gene in the genome of Vibrio and the intragenomic heterogeneity among different copies might be higher than intergenomic heterogeneity in some strains.These facts make the 16S rRNA gene not suitable for the discrimination of V.harveyi group species.Both of single-copy genes toxR and pyrH show a considerable gap between the maximal interspecies similarity and the minimal intraspecies similarity.The toxR gene similarity analyses of eighteen isolates and five reference strains can clearly group them into four species.The phylogenetic tree based on toxR gene shows that strains of individual species clustered together independently,and there are two different clusters within V.alginolyticus,indicating that they have different evolving paths.The similarity and phylogenetic analyses based on pyrH gene come to the same result of toxR gene.The discriminatory power of pyrH gene is slightly lower than that of toxR gene.VITEK 2 COMPACT GN card presents one misidentification among 9 isolates of V.alginolyticus and its species database does not include V.campbellii,V.rotiferianus and V.natriegens.We suggested that toxR gene could be the first choice for fast identification of V.alginolyticus isolates,and pyrH gene may be used for verification.