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The food-borne trematodes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis, have long been recognized as the cause of major human health problems, with an estimated 40 million infected persons. Of the three species of liver fluke, only O. viverrini is classified as a type 1 carcinogen because of its role as an initiator of chronic inflammation and the subsequent development of cholangiocarcinoma. At present, there are no techniques for the early diagnosis of cholangiocarcinoma and it is fatal for most patients. There is considerable variation in parasite prevalence and disease presentation in different geographical areas, the latter of which may be associated with genetic differences among parasites. In the present study, multilocus enzyme electrophoresis was used to provide a comprehensive genetic characterization of O. viverrini from different geographical localities in Thailand and the Peoples’ Democratic Republic of Laos. Parasites from different localities were compared genetically at 32 enzyme loci. The results of the genetic analyses are sufficient to reject the null hypothesis that O. viverrini represents a single species. Therefore, O. viverrini consists of at least two genetically distinct, yet morphologically similar (i.e. cryptic) species. Moreover, there was also separation of the different populations of snails (i.e. the first intermediate hosts) into two distinct genetic groups that corresponded with the delineation of O. viverrini into two species. This suggests that there may be a history of co-evolution in this host–parasite lineage. Additionally, five distinct genetic groups of parasites were detected, each of which occurred within a different and independent river wetland system. Our findings have major implications for the implementation of effective control and surveillance programs targeted to these medically important food-borne parasites.