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Some taxonomic groups are less amenable to mitochondrial DNA barcoding than others. Due to the paucity of molecular information of understudied groups and the huge molecular diversity within flatworms, primer design has been hampered. Indeed, all attempts to develop universal flatworm-specific COI markers have failed so far. We demonstrate how high molecular variability and contamination problems limit the possibilities for barcoding using standard COI-based protocols in flatworms. As a consequence, molecular identification methods often rely on other widely applicable markers. In the case of Monogenea, a very diverse group of platyhelminth parasites, and Rhabdocoela, representing one-fourth of all free-living flatworm taxa, this has led to a relatively high availability of nuclear ITS and 18S/28S rDNA sequences on GenBank. In a comparison of the effectiveness in species assignment we conclude that mitochondrial and nuclear ribosomal markers perform equally well. In case intraspecific information is needed, rDNA sequences can guide the selection of the appropriate (i.e. taxon-specific) COI primers if available. Walter A. Boeger (Universidade Federal do Parana, Brazil), Thierry Backeljau, Marc De Meyer and Kurt Jordaens (Joint Experimental Molecular Unit, Royal Belgian Institute of Natural Sciences/Royal Museum for Central Africa, Belgium), Filip A. M. Volckaert (University of Leuven, Belgium) and Niels Van Steenkiste (Hasselt University, Belgium/Fisheries and Oceans Canada) are gratefully acknowledged for their input into this research. We thank Gontran Sonet (Royal Belgian Institute of Natural Sciences, Belgium) for providing some of the R-scripts and the anonymous reviewers who commented on this manuscript. T.H. was, at the time of writing, a post-doctoral fellow of the Research Foundation - Flanders (FWO-Vlaanderen). M.P.M.V. was supported by KU Leuven - VES/12/005 and by Research Programme G.0553.10 of the Research Foundation - Flanders, and is currently funded by Czech Science Foundation project no. P505/12/G112 (ECIP - Centre of excellence). This research received support from the SYNTHESYS Project (http://www.synthesys.info/) which is financed by European Community Research Infrastructure Action under the FP7 Integrating Activities Programme. Diplectanid molecular analyses were supported by the "Service de Systematique Moleculaire" of the Museum national d'histoire naturelle (CNRS UMS 2700) and the network "Bibliotheque du Vivant" funded by the CNRS, the Museum national d'histoire naturelle, the INRA and the CEA (Genoscope).