ABSTRACT The increase of Vibrio infections especially associated with the consumption of contaminated seafood and fish underlines the necessity of an efficient monitoring system for Vibrio spp. supported by appropriate detection tools both in food products and in clinical samples. The aim of this study was the evaluation of the reliability of the collagenase‐targeted multiplex‐polymerase chain reaction (m‐PCR) for the detection of Vibrio alginolyticus, Vibrio cholerae and Vibrio parahaemolyticus in shellfish samples enriched in alkaline peptone water (APW). The coupling of a pre‐enrichment phase of the samples with the specificity of the PCR‐based assay was applied successfully to the detection of V. alginolyticus and V. parahaemolyticus, demonstrating that collagenase‐target m‐PCR may be used as a valid molecular target to discriminate the three Vibrio species.