Culturable vibrios were isolated from seawater collected during an annual sampling study performed along the Gulf of Trieste coast (Northern Adriatic Sea), and conventional culturing and identification methods were used to investigate the presence of Vibrio parahaemolyticus. Biochemically selected Vibrio strains were subjected to phenotypical identification performed using Alsina's scheme, API 20E and API 20NE. PCR and sequence analysis of the 16S rRNA gene and detection of the species-specific toxR and tlh genes were carried out on strains presumptively identified as V. parahaemolyticus and on a set of unidentified strains to confirm biochemical characterizations. In addition, PCR assays targeting the virulence genes, tdh and trh, were carried out to detect pathogenic strains. PCR results were compared with phenotypic characterizations to evaluate the accuracy of the biochemical methods applied. False-negative identifications were obtained by all phenotypic-based procedures, while API 20E yielded only one false positive. Because the amplification of the 16S rRNA gene produced uncertain results, toxR and tlh gene detections were necessary to confirm the biochemical identifications. Finally, molecular characterization demonstrated the presence of V. parahaemolyticus trh-positive strains and underlined the difficulty in the recognition of the pathogenic environmental organism using conventional methods.