Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community-acquired gastroenteritis and traveller's diarrhoea, we developed a duplex species-specific PCR assay. Methods and Results: Full-length of the 16S-23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR-based sequencing. Two species-specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species-specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2.1 x 10(-2) ng mu l(-1) genomic DNA which corresponds to 5000 CFU ml(-1). Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species-specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.