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Abstract: The use of diagnostic polymerase chain reaction (PCR) is a valuable approach to study host–parasitoid interactions. It circumvents problems inherent to rearing parasitoids from field-collected hosts or host dissection. Here, we present a PCR-based detection method for Diadegma semiclausum, Cotesia glomerata, and Cotesia rubecula, which are key-parasitoids of the three lepidopteran cabbage pests Plutella xylostella, Pieris brassicae, and Pieris rapae, respectively. Primer pairs for the three parasitoid species and Pl. xylostella were developed: they were used either separately in singleplex PCR or combined in multiplex PCR to (1) screen simultaneously for the two Cotesia species or to (2) detect the parasitoid D. semiclausum and identify its host Pl. xylostella in one PCR. The new idea to simultaneously identify parasitoid and host by molecular markers is useful when the host, in our case early-instar larvae of Pl. xylostella, is morphologically difficult to distinguish from other host species also occurring in the same habitat. Concentration–response trials revealed comparable detection sensitivity of singleplex and multiplex PCR, with detection limits ranging from 0.03 to 2.2pg of parasitoid DNA/μl PCR. Furthermore, the different developmental time of immature D. semiclausum and C. glomerata did not influence parasitoid detection success in either assay type. Based on multiplex PCR screening of field-collected caterpillars, we found in Pl. xylostella, P. brassicae, and P. rapae parasitism rates of 33.4% by D. semiclausum, 52% by C. glomerata, and 53.4% by C. rubecula, respectively. [Copyright &y& Elsevier]