The majority of moths are terrestrial throughout their life cycle. An exception is the subfamily Acentropinae (Lepidoptera: Crambidae), a diverse group of aquatic moths comprising nearly 700 species. Acentropinae represent the largest lineage of moths with at least one life stage adapted to an aquatic environment. Despite their unique biology, their evolutionary relationships remain poorly understood. The most comprehensive study on acentropine phylogeny is over 70 years old and predates the use of modern analytical methods. Few studies since then have attempted to reconstruct the phylogeny of Acentropinae, and those that did are limited in taxon and character/gene sampling. We tested hypotheses of acentropine relationships by reconstructing a genus-level phylogeny based on 360 loci and 22 genera of Acentropinae. Our phylogeny provides strong support for the monophyly of Acentropinae, a basal division between two tribes, Argyractini Lange, 1956, and Acentropini Stephens, 1836. The tribe Nymphulini Duponchel is found to be a junior synonym of Acentropini, syn. nov.. Within this tribe, we find support for some previously defined family-group clades (the “terrestrial” clade, the “flowing water” clade, and the Aulacodes clade), and we discuss novel morphological features that are potential synapomorphies of these clades. Our study provides a foundation for future research on the ecology and evolution of aquatic Lepidoptera.