In this study, we describe the development of a fast and accurate molecular identification system for human-associated liver fluke species (Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) using the PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region. Based on sequence variation in the target rDNA region, we selected three species-specific restriction enzymes within the ITS1 regions, generating different restriction profiles among the species: MunI for O. viverrini, NheI for O. felineus, and XhoI for C. sinensis, respectively. Each restriction enzyme generated different-sized fragments specific to the species examined, but no intraspecific polymorphism or cross-reaction between the species was detected in their restriction pattern. These results indicate that PCR-linked restriction analysis of the ITS1 region allows for the rapid and reliable molecular identification among these opisthorchid taxa. In addition, phylogenetic analysis of rDNA sequences using different methods (MP, ML, NJ, and Bayesian inference) displayed O. viverrini and O. felineus as a sister group, but this relationship was not strongly supported. The failure of recovering a robust phylogeny may be due to the relatively small number of synapomorphic characters shared among the species, yielding weak phylogenetic signal. Alternatively, rapid speciation within a very short period time could be another explanation for the relatively poorly resolved relationships among these species. Our data are insufficient for discriminating between sudden cladogenesis and other potential causes of poor resolution. Further information from independent loci might help resolve this phylogeny.