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Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species and biochemical identification requires two or more days to complete. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. However, its accuracy depends on the number of strains in a database. In the current study, we extend and apply the Vibrio database based on MALDI-TOF MS. A total of 74 strains of Vibrio representing 28 species were identified and included in new database. A phylogenetic tree based on rpoB sequence and dendrograms were constructed. We analyzed 30 clinical Vibrio of three species to evaluate the new database and carried out PCA dendrogram analyses for differences of strains. We created a new database that offered fast and accurate Vibrio identification. MSP and PCA dendrogram analyses provided technical support to track sources and incidences of Vibrio infection. In addition, the discovery of characteristic and differential peaks is useful for the future identification of Vibrio. This represents a powerful tool for the rapid and accurate classification and identification of Vibrio and closely related species.