Abstract: Objective To establish a multiplex PCR assay for the rapid detection of pandemic group of Vibrio parahaemolyticus and toxic genes based on GS-PCR. Methods Primers were designed based on the sequences of three genes, including one group-specific gene, toxRS, and two toxic genes, thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). The specificity and sensitivity of the assay were tested with reference strains of pandemic and non-pandemic V.parahaemolyticus strains, and re-identification of 224 food borne strains and 3 clinical isolates of V.parahaemolyticus was conducted. Results The toxRS and tdh were successfully amplified from the pandemic clone of V.parahaemolyticus. tdh were amplified from some clinical isolates of V.parahaemolyticus, while no amplicon was detected in the other bacteria. Detection limits of the assay was 105CFU/ml. Of the 224 food borne isolates and 3 clinical isolates, 5 were identified as pandemic clone, 3 were tdh positive and trh negative, two were tdh negative and trh negative. Conclusion The established multiplex PCR assay is specific and sensitive for the simultaneous detection of pandemic group of V.parahaemolyticus and related toxic genes, which could provide sufficient support for the large scale surveillance for pandemic group of V.parahaemolyticus.