The invasive spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has become a serious pest in the United States. Identification of immature and poorly preserved specimens can be difficult. A molecular diagnostic method for distinguishing D. suzukii from other Drosophila spp. associated with fruit in the United States was developed. A 709-bp region of the mitochondrial DNA cytochrome oxidase I gene was amplified from D. suzukii collections in the United States and compared with sequences of other Drosophila taxa from GenBank. Based on DNA sequence polymorphisms, a polymerase chain reaction—restriction fragment length polymorphism analysis using the restriction enzyme Msp-I was found to differentiate D. suzukii from other Drosophila spp. in the United States. This technique can identify field-collected specimens from various sources and specimens regardless of life stage. This molecular diagnostic method will be useful for monitoring the spread of this economically important invasive insect.