Ophion Fabricius is a diverse genus of nocturnal ichneumonid wasps (Insecta: Hymenoptera) that is particularly species-rich in temperate areas, yet has received little taxonomic attention in the Holarctic region, where most species occur. While there have been some attempts to divide Ophion into monophyletic species groups, the vast majority of species have been lumped into a single, paraphyletic group, the O. luteus species group, which is defined only by the lack of characters specific to the other groups. The challenging morphology of this large catch-all group has limited attempts to subdivide it, and no phylogenetic hypothesis has been proposed for the genus as a whole. In this study, we use DNA sequence data [28S ribosomal RNA (28S), cytochrome oxidase 1 (COI) and internal transcribed spacer 2 (ITS2)] to present the first molecular phylogeny of Ophion. We also describe the secondary structure of ITS2 for the first time in Ichneumonidae, and explore its implications for phylogeny estimation. We define 13 species groups, nine of which were previously considered part of the O. luteus species group s.l. The included species groups are the O. minutus, O. areolaris, O. scutellaris, O. flavidus, O. parvulus, O. slossonae, O. nigrovarius, O. pteridis, O. luteus s.s., and O. obscuratus species groups, along with three groups lacking described species (Species group 1, New Zealand group, Madagascar group). This study provides a framework for future studies of this diverse and morphologically challenging genus.