Lampsiline bivalves typically are considered a tribe within the Unionidae (Ambleminae: Lampsilini), and they display extraordinary morphological adaptations for reproduction. Recent studies have weakly corroborated the monophyly of the Lampsilini, but evolutionary relationships within the tribe and its sister lineage have yet to be elucidated convincingly. However, these determinations are necessary to better understand the evolution of the spectacular morphological diversity present in lampsilines, as well as the specific circumstances surrounding the group's origin. To clarify these matters, phylogenetic analyses were carried out on 2,310 nucleotide and 770 amino acid position matrices containing sequences from five protein coding gene regions on the F and M mitochondrial genomes from 21 amblemine species. Nodal support values on the best Bayesian inference tree robustly confirm the monophyly of lampsilines and a clade containing the following well-supported relationships: (((lampsilines, Popenaias) Plectomerus) Amblema). Furthermore, a maximum likelihood estimate of ancestral character states indicates that the ectobranchy observed in lampsilines Popenaias is homologous and was derived from a tetragenous ancestral lineage. The sister taxon status of P. popeii to the traditional lampsiline taxa and the occasional use of the inner demibranchs for brooding suggest that this species could still retain character states of the lampsiline ancestral lineage. Therefore, additional studies of morphology, reproduction, phylogeography and ecology for Popenaias, Amblema, Plectomerus, and other taxa within the Amblemini could clarify the circumstances surrounding the origin of the lampsiline bivalves.