Carefully collected molecular data and rigorous analyses are revolutionizing today’s phylogenetic studies. Although molecular data have been used to estimate various invertebrate phylogenies for more than a decade, this study is the first survey of different regions of mitochondrial DNA in isopod crustaceans assessing sequence divergence and hence the usefulness of these regions to infer phylogeny at different hierarchical levels. I evaluate three loci from the mitochondrial genome (two ribosomal RNAs (12S, 16S) and one protein-coding (COI)) for their appropriateness in inferring isopod phylogeny at the suborder level and below. The patterns are similar for all three loci with the most speciose suborders of isopods also having the most divergent mitochondrial nucleotide sequences. Recommendations for designing an order- or suborder-level molecular study in previously unstudied groups of Crustacea would include: (1) collecting a minimum of two-four species or genera thought to be most divergent, (2) sampling across the group of interest as equally as possible in terms of taxonomic representation and the distribution of species, (3) surveying several genes, and (4) carrying out preliminary alignments, checking data for nucleotide bias, transition/transversion ratios, and saturation levels before committing to a large-scale sequencing effort.