Vibrio parahaemolyticus is a bacterial pathogen causative of food-borne gastroenteritis. Whole-genome sequencing of V. parahaemolyticus strain RIMD2210633, which exhibits Kanagawa phenomenon (KP), revealed the presence of two sets of the genes for the type III secretion system (T3SS) on chromosomes 1 and 2, T3SS1 and T3SS2, respectively. Although T3SS2 of the RIMD2210633 strain is thought to be involved in human pathogenicity, i.e., enterotoxicity, the genes for T3SS2 have not been found in trh-positive (KP-negative) V. parahaemolyticus strains, which are also pathogenic for humans. In the study described here, the DNA region of approximately 100 kb that surrounds the trh gene of a trh-positive V. parahaemolyticus strain, TH3996, was sequenced and its genetic organization determined. This revealed the presence of the genes for a novel T3SS in this region. Animal experiments using the deletion mutant strains of a gene (vscC2) for the novel T3SS apparatus indicated that the T3SS is essential for the enterotoxicity of the TH3996 strain. PCR analysis showed that all the trh-positive V. parahaemolyticus strains tested possess the novel T3SS-related genes. Phylogenetic analysis demonstrated that although the novel T3SS is closely related to T3SS2 of KP-positive V. parahaemolyticus, it belongs to a distinctly different lineage. Furthermore, the two types of T3SS2 lineage are also found among pathogenic Vibrio cholerae non-O1/non-O139 strains. Our findings demonstrate that these two distinct types are distributed not only within a species but also beyond the species level and provide a new insight into the pathogenicity and evolution of Vibrio species.