Aims: Two well-characterized Vibrio parahaemolyticus pathogenicity factors -thermostable direct haemolysin (TDH) and TDH-related haemolysin - are produced by strains containing the tdh and trh genes, respectively. Most strains of V. parahaemolyticus contain two nonredundant type III secretion systems (T3SS), T3SS1 and T3SS2, both of which contribute to pathogenicity. Furthermore, a recent study has revealed two distinct lineages of the V. parahaemolyticus T3SS2: T3SS2 alpha and T3SS2 beta. The aim of this study was to determine the incidence of these pathogenicity factors in environmental isolates of V. parahaemolyticus. Methods and Results: We collected 130 V. parahaemolyticus isolates (TCBS agar) containing tdh and/or trh (determined by colony hybridization) from sediment, oyster and water in the northern Gulf of Mexico and screened them and 12 clinical isolates (PCR and agarose gel electrophoresis) for pathogenicity factors tdh, trh, T3SS1, T3SS2 alpha and T3SS2 beta. The majority of potential pathogens were detected in the sediment, including all tdh-/trh+ isolates. T3SS2 alpha components were detected in all tdh+/trh- isolates and zero of 109 trh+ isolates. One T3SS2 alpha gene, vopB2, was found in all tdh+/trh- clinical strains but not in any of the 130 environmental strains. Fluorescence in situ hybridization adapted for individual gene recognition (RING-FISH) was used to confirm the presence/absence of vopB2. T3SS2 beta was found in all tdh-/trh+ isolates and in no tdh+/trh- isolates. Conclusions: The combination of haemolysins found in each isolate consistently corresponded to the presence and type of T3SS detected. The vopB2 gene may represent a novel marker for identifying increased virulence among strains. Significance and Impact of the Study: This is the first study to confirm the presence of T3SS2 beta genes in V. parahaemolyticus strains isolated from the Gulf of Mexico and one of the few that examines the distribution and co-existence of tdh, trh, T3SS1, T3SS2 alpha and T3SS2 beta in a large collection of environmental strains.