Several larval parasitoid species are natural enemies of the tortricid moths of European vineyards, including the most damaging of these pests, Lobesia botrana. Over the last few years, DNA-based methods have been used for more rapid and accurate detection and identification of parasitoids. In this study, we developed a PCR-RFLP analysis method targeting a mitochondrial cytochrome oxidase I gene fragment after digestion with the restriction enzyme ApoI, for discrimination between four parasitoid species of Lobesia botrana: Campoplex capitator, Exochus tibialis, Elachertus spp. (Hymenoptera, Ichneumonidae) and Phytomyptera nigrina (Diptera, Tachinidae). We assessed the accuracy of this method using populations of L. botrana sampled from eight vineyards located in South-West of France. On a total of 547 L. botrana larvae collected, 252 were analyzed for parasitism using our molecular method whereas the remaining 295 were reared to assess parasitism rates based on emergence. Our PCR-RLFP method showed a mean parasitism rate of 25%, with values ranging from 3% to 50% across vineyards. The levels of parasitism estimated by this method were about three times those estimated after emergence and identification (7.3%). This difference suggests that mortality may occur during parasitoid development, possibly due to encapsulation. Our method revealed that the two dominating parasitoid species were Campoplex capitator (90%) and Phytomyptera nigrina (9%), whereas the emergence of parasitoids found only C. capitator after taxonomical identification. This study revealed that the PCR-RFLP analysis is an appropriate and reliable tool for estimating the biological control potential of a diverse community of parasitoids on the main tortricid moth of grapevine.