Spotted-wing Drosophila (Drosophila suzukii) is widespread throughout all the important production regions in the world and originates from Asia. In the Russian territory due to strong quarantine and preventive protocols, there are no reports of this fruit fly. Between several methods of the identification of D. suzukii, real-time PCR is a much sufficient method that we used in this project. The proposed method for differentiation is to use the mitochondrial DNA cytochrome oxidase I gene region 709-bp. Performance characteristics of this qualitative assay, validation, and applicability in the All-Russian Plant Quarantine Center are discussed. As a result, we developed, and validated a rapid, highly sensitive and specific primer (3. Dsuz) based on Real-time PCR.