Abstract: 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.