Abstract— Oenothera sect. Pachylophus has proven to be a valuable system in which to study plant-insect coevolution and the drivers of variation in floral morphology and scent. Current species circumscriptions based on morphological characteristics suggest that the section consists of five species, one of which is subdivided into five subspecies. Previous attempts to understand species (and subspecies) relationships at a molecular level have been largely unsuccessful due to high levels of incomplete lineage sorting and limited phylogenetic signal from slowly evolving gene regions. In the present study, target enrichment was used to sequence 322 conserved protein-coding nuclear genes from 50 individuals spanning the geographic range of Oenothera sect. Pachylophus , with species trees inferred using concatenation and coalescent-based methods. Our findings concur with previous research in suggesting that O. psammophila and O. harringtonii are nested within a paraphyletic Oenothera cespitosa . By contrast, our results show clearly that the two annual species ( O. cavernae and O. brandegeei ) did not arise from the O. cespitosa lineage, but rather from a common ancestor of Oenothera sect. Pachylophus . Budding speciation as a result of edaphic specialization appears to best explain the evolution of the narrow endemic species O. harringtonii and O. psammophila . Complete understanding of possible introgression among subspecies of O. cespitosa will require broader sampling across the full geographical and ecological ranges of these taxa.