Background: The superfamily Pterioidea is a morphologically and ecologically diverse lineage of epifaunal marine bivalves distributed throughout the tropical and subtropical continental shelf regions. This group includes commercially important pearl culture species and model organisms used for medical studies of biomineralization. Recent morphological treatment of selected pterioideans and molecular phylogenetic analyses of higher-level relationships in Bivalvia have challenged the traditional view that pterioidean families are monophyletic. This issue is examined here in light of molecular data sets composed of DNA sequences for nuclear and mitochondrial loci, and a published character data set of anatomical and shell morphological characters. Results: The present study is the first comprehensive species-level analysis of the Pterioidea to produce a well-resolved, robust phylogenetic hypothesis for nearly all extant taxa. The data were analyzed for potential biases due to taxon and character sampling, and idiosyncracies of different molecular evolutionary processes. The congruence and contribution of different partitions were quantified, and the sensitivity of clade stability to alignment parameters was explored. Conclusions: Four primary conclusions were reached: (1) the results strongly supported the monophyly of the Pterioidea; (2) none of the previously defined families (except for the monotypic Pulvinitidae) were monophyletic; (3) the arrangement of the genera was novel and unanticipated, however strongly supported and robust to changes in alignment parameters; and (4) optimizing key morphological characters onto topologies derived from the analysis of molecular data revealed many instances of homoplasy and uncovered synapomorphies for major nodes. Additionally, a complete species-level sampling of the genus Pinctada provided further insights into the on-going controversy regarding the taxonomic identity of major pearl culture species.