Foodborne disease caused by Vibrio parahaemolyticus is a serious public health problem in many countries. Molecular typing has a great scientific significance and application value for epidemiological research of V. parahaemolyticus. In this study, a real-time PCR with melting curve analysis was established for molecular typing of V. parahaemolyticus. Eighteen large variably presented gene clusters (LVPCs) of V. parahaemolyticus which have different distributions in the genome of different strains were selected as targets. Primer pairs of 18 LVPCs were distributed into three tubes. To validate this newly developed assay, we tested 53 Vibrio parahaemolyticus strains, which were classified in 13 different types. Furthermore, cluster analysis using NTSYS PC 2.02 software could divide 53 V. parahaemolyticus strains into six clusters at a relative similarity coefficient of 0.85. This method is fast, simple, and conveniently for molecular typing of V. parahaemolyticus.