The central clade of the genus Vibrio, also called the Vibrio core group, comprises six species that are tightly related (DNA–DNA reassociation values are very close to 70 % for most species pairs). Identification of novel strains to the species level within this group is troublesome and results are quite often dependent on the methodology employed. Therefore, this group represents an excellent framework to test the robustness of multilocus sequence analysis (MLSA) not only for inferring phylogeny but also as an identification tool without the need for DNA–DNA hybridization assays. The genes selected, 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR, were amplified by direct PCR from 44 Vibrio core-group strains. Subsequent analysis allowed us to recognize toxR and rpoD as the most resolving individual genes and showed that concatenated sequences of rpoD, rctB and toxR were more useful than concatenated sequences of all seven genes. To validate our conclusions, MLSA similarities have been correlated with DNA–DNA relatedness values obtained in this study and values taken from the literature. Although the seven concatenated genes gave the best correlation, the concatenated sequences of rpoD, rctB and toxR have the practical advantage of showing a considerable gap between the maximal interspecies similarity and the minimal intraspecies similarity recorded, meaning that they can be used quite conveniently for species identification of vibrios.