Freshwater mussels (Bivalvia, Unionidae) have suffered strong declines over the last century. High morphological plasticity of Unionidae causes disturbances in their systematics and taxonomy, hampering conservation efforts. Species that have historically been placed under the North American genus Quadrula have suffered from numerous taxonomic and species delineation problems since its inception. Four genera are presently recognized within Quadrula sensu lato, that is, Cyclonaias, Quadrula, Theliderma and Tritogonia, but their phylogenetic basis remains incompletely tested. In the present study, we reconstructed several two-marker (mtDNA cytochrome c oxidase subunit I-COI and NADH dehydrogenase subunit 1-ND1) phylogenies with newly collected specimens and all previously available sequences covering most species within this group. We then delineated the species within the group using an integrative approach with the application of molecular statistical methods, morphometric (Fourier Shape) analyses and geographic distribution data.Four clades corresponding to these genera were consistently recovered in all phylogenies. To validate the generic status of these clades, molecular analyses were complemented with morphological, anatomical and ecological data compiled from the literature. Several revisions are here proposed to the current systematics and taxonomy of these genera, including the synonymization of Cyclonaias asperata under Cyclonaias kieneriana; the inclusion of Quadrula apiculata and Quadrula rumphiana under Quadrula quadrula; the placement of Quadrula nobilis under Tritogonia; and finally the separation of the Mobile River basin populations of Theliderma metanevra as a new species, that is, Theliderma johnsoni n. sp. The conservation implications of the proposed changes are then discussed. 314 | LOPES-LIMA Et AL. F I G U R E 1 Map of all sampling sites for the present study; both tissue and shell materials in red; only shell materials in white [Colour figure can be viewed at wileyonlinelibrary.com] F I G U R E 2 Bayesian consensus tree inferred from the cytochrome c oxidase subunit I (COI) gene fragment. The values above and below the nodes indicate Bayesian posterior probability (bpp) percentage and maximum likelihood bootstrap values (bs), respectively. Values over 95% are represented by an asterisk, and those <50% are not shown for clarity [Colour figure can be viewed at wileyonlinelibrary.com] SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Lopes-Lima M, Burlakova L, Karatayev A, et al. Revisiting the North American freshwater mussel genus Quadrula sensu lato (Bivalvia Unionidae): Phylogeny, taxonomy and species delineation. Zool Scr. 2019;48:313-336. https://doi.