Human opisthorchiais caused by the liver fluke Opisthorchis viverrini is a major fish-borne trematode infection endemic in the Southeast Asian countries. The infection is acquired through consumption of raw fish harboring metacercariae of O. viverrini. Owing to potential risk of transmission of opisthorchiasis through fish trade, rapid and reliable detection methods have gained importance to ensure food safety. In the study described here, we report successful development and evaluation of a polymerase chain reaction (PCR) assay for the detection of O. viverrini, based on the nucleotide sequence derived in this study. The assay is specific with no cross-reaction with other trematodes commonly found in fish including the closely related species, Clonorchis sinensis. The sensitivity of the assay was determined to be 10(-12)ng of O. viverrini DNA while in artificially spiked fish meat, 3 metacercariae could be detected. The results suggest that the PCR method described here is specific to O. viverrini with potential application in fish quarantining.