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Among others, the families Opisthorchiidae and Heterophyidae includes several genera causing fish-borne zoonoses and distributed also in European Countries and that are included in the ParaFishControl (Advanced Tools and Research Strategies for Parasite Control in European farmed fish) H2020 EU project. Due to the small size of the metacercariae, the infective stage for human, these parasites cannot be detected visually in fish and monitoring requires expert application of time-consuming techniques. The aim of this was to develop a rapid and affordable molecular method based on multiplex PCR for simultaneous identification of metacercariae of the most common European Opisthorchiid and Heterophyid in fish or fish products.