The barnacles of the superorder Acrothoracica are small, burrowing, epibiotic, and dioecious (large female with dwarf male) crustaceans largely found in the carbonate sediments and skeletons of marine invertebrates. The acrothoracicans represent the Cirripedia with the most plesiomorphic characters and have prominently featured in phylogenetic speculations concerning these crustaceans. Traditionally, Acrothoracica was divided into two main orders, Pygophora and Apygophora. The Apygophora had uniramus cirri and no anus. The Pygophora had biramus terminal cirri and an anus and was further divided into two families, Lithoglyptidae and Cryptophialidae. Kolbasov (2009) revised the superorder Acrothoracica on the basis of morphological examinations of females, dwarf males, and cyprids and rearranged the acrothoracican species into two new orders, Lithoglyptida and Cryptophialida. The present study is the first attempt to reconstruct the phylogenetic relationships of acrothoracican barnacles by sequencing two mitochondrial (cytochrome C oxidase I and 16S ribosomal DNA) and two nuclear (18S ribosomal DNA and histone H3) markers of 8 of the 11 genera comprising 23 acrothoracican species. All monophylies of the eight acrothoracican genera sampled in this study were strongly supported. The deep interfamilial relationship constructed is consistent with the recent morphological phylogenetic relationship proposed by Kolbasov, Newman, and Høeg (Kolbasov, 2009) that Cryptophialidae (order Cryptophialida) is the sister group to all other acrothoracicans (order Lithoglyptida). According to an ancestral character state reconstruction analysis, the posterior lobes of females; armament of opercular bars, attachment stalk, lateral projections of the body, and aperture slits in dwarf males; and habitat use appear to have phylogenetic importance.