The aim of this study was to develop TaqMan real-time PCR assay that detected Opisthorchis viverrini DNA from 18 normal and 18 tumor tissue specimens from Thai patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), who underwent liver resection from October 2005 to May 2006. Control liver specimens were seven non-primary liver cancers. A conserved probe representing 100% sequence homology was used as a reference for O. viverrini-specific probe. Five of six tumors (83%) and all six normal tissues from CCA group; and seven of twelve tumors (58%) and ten of twelve normal tissues (83%) from HCC group were found to have O. viverrini DNA. The O. viverrini DNA detection among HCC and CCA patients were not associated (p=0.193; 90%CI). This RT-PCR will be a useful tool for investigating the relationship between cancer type and presence of the parasite and also for conducting epidemiological surveys.