Hymenopteran parasitoids generally show a haplo-diploid sex determination system. Haploid males are produced from unfertilized eggs, whereas diploid females develop from fertilized eggs (arrhenotokous). In some cases, diploid females develop from unfertilized eggs (thelytokous). Diglyphus wani (Hymenoptera: Eulophidae) is a biological control agent for agromyzid leafminers and have arrhenotokous and thelytokous strains. However, the morphological characteristics of two strains of D. wani are so similar that it is difficult to accurately distinguish them based on morphology. Here, a rapid molecular identification method was developed based on the mitochondrial gene cytochrome c oxidase I (COI) and one-step multiplex PCR. Two primer combinations, PC1 (Ar-F1/Th-F1/WR2) and PC2 (Ar-F1/Th-F4/WR2), were designed and repeatedly screened to distinguish two strains simultaneously, of which two special forward primers Th-F1/Th-F4 were used for the thelytokous strain and one special forward primer Ar-F1 was used for the arrhenotokous strain. In addition, a common reverse primer, WR2, was used for both strains. The PC1 and PC2 PCR assays were effective in distinguishing the two strains at different developmental stages and field colonies. This method provides a reliable, highly sensitive, and cost-effective tool for the rapid identification of the two strains of D. wani.