We reconstructed the phylogenetic relationships among 37 species representing seven main lineages within Opisthobranchia (Mollusca: Gastropoda) based on a mitochondrial fragment that included partial cox 1, complete trnV, and partial rrnL genes (about 2500 bp). Phylogenetic analyses confirmed tentatively that all studied main opisthobranch lineages conformed monophyletic groups except Nudibranchia. The sacoglossan Ascobulla was placed as the most basal lineage of opisthobranchs. The basommatophoran pulmonate Siphonaria was recovered within Opisthobranchia between Ascobulla and the remaining opisthobranchs. The latter were divided into two different lineages that await formal description: on one side, Cephalaspidea, Tylodinoidea, and Anaspidea (sharing features in the reproductive, digestive, and circulatory systems) were grouped together and, on the other Architectibranchia and Nudipleura (sharing similarities in the circulatory system) were recovered as sister group taxa. Two well-supported clades were recovered within Nudipleura: Pleuroanthobranchia (new taxon) and Cladobranchia. Pleuroanthobranchia (Pleurobranchoidea plus Anthobranchia) was defined by the presence of blood gland, the presence of calcareous spicules in the integument and the presence of a caecum with an opening directly into the stomach. The new molecular phylogeny provided a robust framework for comparative studies, and prompted a revision of the morphological synapomorphies diagnosing the main clades within opisthobranchs.