Aims: To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus. Methods and Results: Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates (n = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10(7)-10(1) CFU ml(-1) and that for tdh2 was 10(7)-10(4) CFU ml(-1). Conclusions: The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min. Significance and Impact of the Study: The assay developed allows accurate detection of pathogenic V. parahaemolyticus, which is valuable for rapid tracing of infection source during outbreaks.