The presence of metacercariae and adults of the trematode Pseudamphistomum truncatum in roach and mink, respectively, was recorded in Lake Fure North of Copenhagen, Denmark. This zoonotic digenean opisthorchiid represents a threat to humans due to its ability to infect the biliary system following ingestion of inadequately processed infected fish. Therefore precise species identification of infective metacercariae in fish used for human consumption is essential. Due to the relatively limited information on metacercarial identity obtained by morphometric studies a series of molecular techniques were used to link the larval parasite in fish with the un-equivocally diagnosed adults in the biliary system of the mink. By the use of carefully selected polymerase chain reaction (PCR) primers and subsequent sequencing of the ITS region from both metacercariae and adults full sequence identity of both metacercariae and adults were confirmed. The presence of this parasitosis in fish from a lake used for both commercial and recreational fisheries call for hygienic alerts in order to prevent accidental human infection with this opisthorchiid.