A phylogenetic analysis of Polycladida based on two partial mitochondrial genes (cox1 and 16S) is provided. The analysis includes 30 polyclad terminals that represent species from the two taxa which traditionally divide the groups Cotylea and Acotylea. Our phylogenetic analyses produced a well-supported hypothesis that confirms the monophyly of Polycladida, as well as Acotylea and Cotylea. Within Acotylea, there are two lineages not highly supported: on one hand, Leptoplanoidea (excluding <i>Hoploplana elisabelloi</i>) and one Stylochoidea member (<i>Pseudostylochus intermedius</i>) (classification sensu Faubel, 1983, 1984), and on the other hand, Stylochoidea members together with <i>Discocelis tigrina</i> and <i>H. elisabelloi</i>. The genera <i>Stylochus</i> and <i>Imogine</i> are not monophyletic. Within Cotylea, Pseudocerotidae and Euryleptidae are monophyletic, though not highly supported, while Prosthiostomidae is not. Euryleptoidea is paraphyletic. The genera <i>Pseudobiceros</i> and <i>Pseudoceros</i> are monophyletic and highly supported. Our results suggest that, within Acotylea, the prostatoid organs of <i>Discocelis</i> may have been derived from a prostatic vesicle. The genus <i>Hoploplana</i> could be included in Stylochoidea. Within Cotylea, the common ancestor of Euryleptidae and Pseudocerotidae might have been an aposematic animal with tentacles.Facultad de Ciencias Naturales y Museo