BACKGROUND: Several foodborne outbreaks associated with the consumption of vegetables have been reported which involved Vibrio spp . as causative agents. Conventional methods of detecting these microorganisms are time-consuming. Therefore, the development of techniques for rapid detection seems to be of paramount importance. OBJECTIVES: The present study recommends a rapid and reliable method for the detection of Vibrio cholera (V. chol-era), V. parahaemolyticus, V. vulnificus, and V. alginolyticus. Moreover, the results are compared with the conventional plate culture method. METHODS: The conventional bacteriological tests were conducted to detect Vibrio spp. in vegetables and their surrounding water. The samples were also subjected to a newly developed multiplex polymerase chain reaction (PCR) using five specific genes, including VC-Rmm of V. cholerae, VP-MmR of V. parahaemolyticus, VV-Rmm of V. vulnificus, V.al2-MmR of V. alginolyticus, and VM-F for all the four isolates. RESULTS: The presence of V. alginolyticus and V. vulnificus was confirmed by amplifying the specific regions of 412 bp for V. vulnificus and 144 bp for V. alginolyticus. The results demonstrated that V. cholerae and V. parahaemolyticus were not detected in multiplex PCR, which was consistent with the findings of conventional plating methods. CONCLUSIONS: Obtained results revealed that the designed multiplex PCR assay is a reliable, rapid, and cost-effective method for the simultaneous detection of Vibrio spp .