Metacercariae, morphologically similar to those of small liver flukes, were found to parasitize red-tailed snakehead fish, Channa limbata, collected from the city of Vientiane, Lao People's Democratic Republic. Adult worms that were recovered from experimentally-infected hamsters showed characteristics distinctly different from Opisthorchis viverrini, but closely similar to Opisthorchis lobatus, which was first reported in poultry (Anas sp.) from Pakistan. The present study aimed to redescribe O. lobatus based on the adult worms recovered from experimentally-infected hamsters. Additionally, it aimed to document the genetic relationships among O. lobatus and other opisthorchiid liver flukes using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and the internal transcribed spacer 2 (ITS2) region. DNA alignment of the O. lobatus and O. viverrini COI partial sequences (330 bp) showed 3.03% fixed differences (2.72% of amino acids changed) while the ITS2 region (350 bp) indicated a 0.86% difference for nucleotides. Species boundaries between the 2 parasites were determined by neighbor-joining analysis using the molecular sequence data. The phenogram confirmed that O. lobatus was distinctly different from O. viverrini, representing the first reported instance of O. lobatus in the Greater Mekong Sub-region (GMS) and the first record of C. limbata as the second intermediate host of a small liver fluke. Questions regarding human infection and the extent of the geographic distribution of these species should be investigated further.