Phylogenetic analysis combined with chemical ecology can contribute to the delimitation of closely related insect species, particularly in Lepidoptera. In this study, the taxonomic status of a species in the genus Busseola (Lepidoptera: Noctuidae) was discussed using morphological data, cross-mating experiments, sex pheromone chemistry, field-trapping, and molecular classification. The results of the chemical ecology experiments corroborated those from the phylogeny studies. It was concluded that several reproductive isolation components, namely host plants, geography, pheromone emission time, pheromone blend, and post-zygotic isolation factors, led to the separation of Busseola n. sp. from its closely related species B. segeta. Molecular data showed a strong difference between these two species, regardless of the marker used. The new species named Busseola nairobica was morphologically described and a hypothesis about the evolutionary history of the studied species was put forward.