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Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafoodrelated illnesses and also can cause wound infections. These bacteria often co-exist in marine and estuarine environments. However, there have been no reported protocols that can detect and characterize (i.e. pathogenic or nonpathogenic) them in a single PCR. In this study, we developed a pPCR assay with a combination of two species-specific and three pathogenic-specific PCR primers to simultaneously detect virulent (viuB in V. vulnificus and tdh/trh in V. parahaemolyticus) and nonvirulent (vvhA in V. vulnificus and tlh in V. parahaemolyticus) markers of the two species in bacterial isolates. The assay was validated by three methods. First, the pPCR was used to characterize 300 bacterial isolates consisting of seven reference strains and 293 environmental strains isolated from the Gulf of Mexico water. Results were compared with characterizations based on single-gene PCR amplifications and previously published multiplex PCR protocols. Second, 51 isolates characterized with the pPCR were analysed by 16S rRNA sequencing to confirm any false-negative/positive reaction. Finally, the effectiveness of the assay for heterogeneous bacterial samples was validated. The pPCR correctly characterized isolates from the Gulf with an efficiency of 96·6–98·7%.Significance and Impact of the Study: This study, to the best of our knowledge, has been the first effort to develop a fast, cost-effective multiplex PCR (a pentaplex PCR termed pPCR) assay for simultaneous detection of bacterial isolates for pathogenic and nonpathogenic strains of Vibrio parahaemolyticus and Vibrio vulnificus. The fact that the assay can be applied to screen and characterize large numbers of bacterial isolates for the two most concerned Vibrio spp. from clinical/environmental samples will help detect pathogens, prevent disease outbreaks and plan better risk management strategies to protect public health.