The diamondback moth, Plutella xylostella (Linnaeus) has a cosmopolitan distribution and is one of the major pests on cruciferous plants. Biological control, especially with species of the genus Diadegma, has been successfully employed in several parts of the world, mainly in South East Asia. The taxonomy of this genus based on classical morphological characters is still unclear and misidentifications are reported. In the present study seven Diadegma species associated with P. xylostella were separated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses. The second internal transcribed spacer (ITS2) of the ribosomal DNA (rDNA) was successfully amplified in all 167 individuals and digested using 11 different restriction enzymes. One restriction enzyme (CfoI) showed different restriction profiles in all species and also between two population samples of D. mollipla (Holmgren) from eastern and southern Africa. In addition, a new Diadegma species associated with P. xylostella from Ethiopia was discovered. Techniques; Genetics; Parasites diseases and disorders; Parasites; Insect parasites; Hosts; Insect hosts Plutella xylostella (Plutellidae); Hymenopteran parasites; Diadegma; Parasite species discrimination using molecular analysis Diadegma (Ichneumonidae); Genetic techniques; Identification techniques; PCR-based approach for distinguishing species associated with lepidopteran host; Molecular genetics, rDNA internal transcribed spacer 2 region; Lepidopteran hosts; Plutella xylostella none