Abstract The quality of a sperm population can be characterized physiologically and its fecundity predicted by its viable : non-viable sperm ratio. To improve the knowledge of reproductive strategies in two ectoparasitoid hymenopteran species, Eupelmus orientalis Crawford (Hymenoptera: Eupelmidae) and Dinarmus basalis Rondani (Hymenoptera: Pteromalidae), the assessment of sperm viability using the dual fluorescence staining procedure SYBR-14 : propidium iodide was developed. The aim of the study was to provide a comparative test in vitro applicable to both sexes to study the evolution of sperm quality at various stages of the reproductive processes. The reliability of propidium iodide to detect non-viable sperm (stained in red) was confirmed in both species on the basis of two stress tests (ethanol and Triton X-100) but our study also revealed that propidium iodide concentrations must be adequately adjusted for each single species. This experiment also demonstrated the physiological heterogeneity of sperm populations in E. orientalis and D. basalis males and females. In both species, 40% of the sperm in the seminal vesicles was found to be non-viable. By contrast with E. orientalis, the populations of non-viable sperm estimated from the seminal vesicles of D. basalis were found to be strongly different from those observed in the spermatheca. From the present results, the population of viable sperm detected in the spermatheca of females from both species proved a reliable predictor of fertilization achieved in ovipositing females.