Accurate phenotypic identification of Vibrio species is problematic, largely because of the great variability in biochemical characteristics. So; the purpose of this study was to investigate the prevalence of Vibrio spp. in shellfish using the biochemical and multiplex PCR methods. This study was conducted on 50 mussels samples collected during summer months of 2011 and 2012. The obtained results indicated that Vibrio species isolated from the examined mussels samples collected from Alexandria and Suez gulf were 36% and 28 %, respectively. All the biochemically Vibrio species positive samples were tested using mPCR in order to identify Vibrio sp. of public health importance as, V. vulnificus, V. cholerae, V. parahaemolyticus and V. mimicus. The results of mPCR in mussel samples were compared with those obtained by biochemical methods and it was concluded that the multiplex PCR allowed us to identify Vibrio species quickly and accurately.