Representatives of the Meidiamidae and Otomesostomidae (Platyhelminthes: Proseriata) are seldom encountered, and the monophyly and phylogenetic relationships of these families have never been assessed on molecular basis. Here, we present the first exhaustive molecular study of Proseriata at the family level, including species belonging to the genera Meidiama and Yorknia (Meidiamidae), and Otomesostoma auditivum (Otomesostomidae), using 18S and 28S genes as markers. We performed phylogenetic analyses (Maximum Likelihood [ML] and Bayesian Inference [BI] methods) and species delimitation methods (Single/Multiple Threshold-Generalized Mixed Yule Coalescent [ST/MT-GMYC] and Poisson Tree Processes [PTP/bPTP]). The taxon Meidiamidae was not supported, since the type species (Meidiama lutheri) and Meidiama etrusca sp. n. are nested within the Archimonocelididae, formerly restricted to specialized cnidarian feeders. Species belonging to the genus Yorknia resulted genetically well separated from species of Meidiama and from the rest of Archimonocelididae. The new family-level taxon Yorkniidae fam. n. is thus here introduced, to include the type species of Yorknia (Yorknia aprostatica), and six new species, five of which are formally described here. Otomesostoma auditivum, representative of Otomesostomidae, the only exclusively freshwater taxon of the Proseriata, is the sister taxon of the predominantly marine Apingospermata. This result is not conflictual with the family level attributed to Otomesostomidae on morphological grounds, but it raises speculations on the marine versus freshwater origin of Apingospermata.