Acotylea is a suborder of Polycladida (Rhabditophora, Platyhelminthes) characterized by lack of a cotyl (sucker-like structure) on the ventral surface of the body. We newly determined partial sequences of two mitochondrial (16S ribosomal RNA and cytochrome c oxidase subunit I) and two nuclear (18S and 28S ribosomal RNA) genes from 24 acotylean species (12 families and 14 genera). Based on these sequences in addition to those available in public databases, we inferred the phylogeny of 16 families and 27 genera of Acotylea from molecular phylogenetic analyses (maximum likelihood and Bayesian inference) based on concatenated gene sequences. Our analyses supported three clades corresponding to Discoceloidea, Leptoplanoidea, and Stylochoidea. The phylogenetic position of Callioplanidae remains unclear. Among family- or genus-level taxa, Gnesiocerotidae, Stylochoplanidae, and Comoplana were not monophyletic. We discuss the validities of Notocomplanidae and Koinostylochus, and the family-level assignment of Mirostylochus.