Trilobed uncus taxa of the genus Ostrinia (Lepidoptera, Crambidae) illustrate the complex relationship, at early stages of speciation, between reproductive isolation and differentiation in morphology, resource use and genetic variation. On the basis of behaviour and ecology, we recently hypothesized that individuals with small mid-tibiae belong to two distinct species depending on host plant-O. nubilalis and O. scapulalis sensu Frolov et al. (2007) feeding on maize and on a number of dicotyledons, respectively. Individuals with small, medium or massive mid-tibiae would all belong to O. scapulalis as long as they feed on these dicotyledons. This contrasts with previous taxonomy, which distinguished three species by male mid-tibia morphology, regardless of host plant. Here, we test our hypothesis by examining the genetic structure of Ostrinia populations from regions with mid-tibia polymorphism Western Russia and Kazakhstan and comparing it with that of French populations where only small mid-tibiae occur. Results support two predictions: (1) maize- and dicotyledon-collected populations are genetically differentiated from each other like in France, and (2) dicotyledon-collected populations show no genetic evidence of consisting of more than one species. Between-species differentiation was unrelated to geographic distance, despite significant isolation by distance within species. The distinction between two and only two species differing by host plant thus holds at continental scale. Interestingly, one microsatellite locus contributed 10 times more than the others to differentiation between both taxa. This deserves further investigation, as it might reveal a linkage between this outlier and loci involved in host-plant adaptation and/or reproductive isolation.