Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2␣ gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections. Vibrio parahaemolyticus is the leading cause of seafood-associated gastroenteritis in the United States and the world (1). The U.S. Food and Drug Administration (1) estimates that 4,500 reported cases of V. parahaemolyticus gastroenteritis occur every year, and outbreaks of V. parahaemolyticus infections are increasing in frequency and expanding in geographic range (2, 3). This organism is ubiquitous in nearshore marine waters, and cell numbers are typically highest in surficial sediments (4) and in turbid waters bearing high loads of resuspended sediment (5, 6). Filterfeeding bivalve mollusks, such as oysters and mussels, can concentrate V. parahaemolyticus and other pathogenic vibrios (for examples, see references 7 and 8), resulting in levels in the mollusks capable of producing infection in a person that ingests them (9). Virulent V. parahaemolyticus strains are clearly a concern for seafood safety, and their detection is important anywhere that elevated levels of this organism are found.Detection of V. parahaemolyticus in shellfish and environmental samples is typically based on molecular biological analysis of specific genes, particularly genes exclusive to this species and those strongly correlated with pathogenicity. The gene encoding the thermolabile hemolysin (TLH), designated tlh, encodes a phospholipase A2 (10). While its contribution to V. parahaemolyticus pathogenicity is unknown, expression of this gene is upregulated under conditions mimicking the human intestine (11,12). tlh is considered to be a species-specific marker for V. parahaemolyticus (13,14) and is frequently employed to identify this species (1,13,(15)(16)(17)(18). Genes encoding the thermostable direct hemolysin (TDH) and the homologous thermostable direct hemolysin-related hemolysin (TRH), tdh and trh, respe...