We apply a molecular approach to quantify the level of hymenopteran parasitoids infestation in the larvae of the marsh fritillary (Euphydryas aurinia), a declining butterfly species, in western Bohemia, Czech Republic, in two subsequent years. We used the novel primer HymR157 in combination with known universal 28SD1F to establish a PCR detection system which amplifies hymenopteran parasitoids, but not the lepidopteran host. In the 14 sampled E. aurinia colonies, the infestation rates per individuum were 33.3% and 40.2%; whereas per sampled larval colony, these were on average 38.5% (range 0–100) and 40.1% (0–78). The per-colony infestation rates correlated with the numbers of larval webs censused per colony the year prior to sampling the parasitoids, pointing to a time lag in parasitoid infestation rates. The levels of the hymenopteran parasitoid prevalence are thus relatively high, supporting the importance of parasitoids for the population dynamics of the threatened host. The detection primers we developed can detect a range of hymenopteran parasitoids on other butterfly hosts.