Two conflicting morphological approaches to polyclad systematics highlight the relevance of molecular data for resolving the interrelationships of Polycladida. In the present study, phylogenetic trees were reconstructed based on a short alignment of the 28S rDNA marker gene with 118 polyclad terminals (24 new) including 100 different polyclad species from 44 genera and 22 families, as well as on a combined dataset using 18S and 28S rDNA genes with 27 polyclad terminals (19 new) covering 26 different polyclad species. In both approaches, Theamatidae and Cestoplanidae were included, two families that have previously been shown to switch from Acotylea to Cotylea. Three different alignment methods were used, both with and without alignment curation by Gblocks, and all alignments were subjected to Bayesian inference and maximumlikelihood tree calculations. Over all trees of the combined dataset, an extended majority-rule consensus tree had weak support for Theamatidae and Cestoplanidae as acotyleans, and also the cotylean genera Boninia, Chromyella and Pericelis appeared as acotyleans.With the most inclusive short 28S dataset, on the other hand, there is good support for the aforementioned taxa as cotyleans. Especially with the short 28S matrix, taxon sampling, outgroup selection, alignment method and curation, as well as model choice were all decisive for tree topology. Well-supported parts of the phylogeny over all trees include Pseudocerotoidea, Prosthiostomoidea, Stylochoidea, Leptoplanoidea and Cryptoceloidea, the latter three with new definitions. Unstable positions in the tree were found not only for Theamatidae, Cestoplanidae, Boninia, Chromyella and Pericelis, but also for Anonymus, Chromoplana and Cycloporus.