Molecular data are used to test whether (1) Phreatoicidea are the earliest derived living isopods, and (2) the long-tailed isopod morphology is the derived condition within the Isopoda. Small and large subunits of the mitochondrial ribosomal genes (12S- and 16S rDNA), and cytochrome oxidase c subunit I (COI) are used as a case study for exploring the boundaries of applicability of these genes at this taxonomic level. I evaluate three data sets, compare three differently weighted alignments, test data partitions for congruence and phylogenetic structure, and evaluate the topologies of individual and combined data partitions. The 12S- and 16S rDNA partitions are not incongruent. However, the incongruence between ribosomal and COI partitions is significant. The study provides new data for addressing generic, familial, and subordinal relationships of this large, morphologically and ecologically diverse taxon. For the three data sets investigated here, the addition of taxa increases bootstrap values at nodes, more nodes have bootstrap support greater than 50%, and clade topologies are comparable when taxa are added. These mitochondrial genes corroborate isopod clades previously recognized on morphological grounds, and in other instances, suggest relationships not previously proposed, i.e., valviferans had a sphaeromatid ancestor, and oniscids and sphaeromatids may be more closely related than previously thought.