Russian wheat aphid (RWA) (Diuraphis noxia Kurdjumov) is a serious invasive pest of small-grain cereals and many grass species. An efficient strategy to defy aphid attacks is to identify sources of natural resistance and transfer resistance genes into susceptible crop cultivars. Revealing the genes helps understand plant defense mechanisms and engineer plants with durable resistance to the pest. To date, more than 15 RWA resistance genes have been identified in wheat (Triticum aestivum L.) but none of them has been cloned. Previously, we genetically mapped the RWA resistance gene Dn2401 into an interval of 0.83 cM on the short arm of chromosome 7D and spanned it with five bacterial artificial chromosome (BAC) clones. Here, we used a targeted strategy combining traditional approaches toward gene cloning (genetic mapping and sequencing of BAC clones) with novel technologies, including optical mapping and longread nanopore sequencing. The latter, with reads spanning the entire length of a BAC insert, enabled us to assemble the whole region, a task that was not achievable with short reads. Long-read optical mapping validated the DNA sequence in the interval and revealed a difference in the locus organization between resistant and susceptible genotypes. The complete and accurate sequence of the Dn2401 region facilitated the identification of new markers and precise annotation of the interval, revealing six highconfidence genes. Identification of Epoxide hydrolase 2 as the most likely Dn2401 candidate opens an avenue for its validation through functional genomics approaches.