Identification and classification of Vibrio species have relied upon band pattern methods (e.g., amplified fragment length polymorphism) and DNA-DNA hybridization. However, data generated by these methods cannot be used to build an online electronic taxonomy. In order to overcome these limitations, we developed the first standard multilocus sequence scheme focused on the ubiquitous and pathogenic Vibrio harveyi species group (i.e., V. harveyi, V. campbellii, V. rotiferianus, and a new as yet unnamed species). We examined a collection of 104 isolates from different geographical regions and hosts using segments of seven housekeeping genes. These two species formed separated clusters on the basis of topA, pyrH, ftsZ, and mreB gene sequences. The phylogenetic picture obtained by the other three loci, i.e., gyrB, recA, and gapA, was more complex though. V. campbellii appeared nested within V. harveyi in the recA trees, whereas V. harveyi formed a tight nested cluster within V. campbellii by gapA. The gyrB gene had no taxonomic resolution and grouped the two species together. The fuzziness observed in these three genes seems not be related to recombination but to low divergence due to the accumulation of only a few substitutions. In spite of this, the concatenated sequences provided evidence that the two species form two separated clusters. These clusters did not arise by recombination but by accumulation of point mutations. V. harveyi and V. campbellii isolates can be readily identified through the open database resource developed in this study (http://www.taxvibrio.lncc.br/). We argue that the species should be defined by evolutionary criteria. Strains of the same species will share at least 95% concatenated sequence similarity using the seven loci, and, most importantly, cospecific strains will form cohesive readily recognizable phylogenetic clades.The species Vibrio harveyi and V. campbellii are widespread in the marine environment and among the main species responsible for disease in many wild and reared aquatic organisms, most notably peneid shrimp, several fish species, and mollusks (2). Luminous vibrios related to V. harveyi have been implicated principally in disease outbreaks in shrimp larviculture facilities and in grow-out ponds worldwide. More recently V. harveyi has been associated with infections in corals (20). Indeed recent studies have shown that vibrios, including the V. harveyi group, are abundant in the mucus of corals and may cause infections during periods of environmental imbalances (15).In the past, the identification of V. harveyi and related species isolated from the marine environment has been imprecise and represents hard work as it involves performing many biochemical and/or physiological tests (14). Presumptive V. harveyi isolates grow on thiosulfate-citrate-bile saltssucrose agar are motile, ferment glucose, and are oxidase positive and sensitive to the vibriostatic agent 0/129 at 150 g. They are arginine dihydrolase negative and lysine and ornithine decarboxylase positive. Most presump...