Many animal species experiencing spatial or interannual fluctuations of their environment are capable of prolonged diapause, a kind of dormancy that extends over more than one year. Such a prolonged diapause is commonly perceived as a temporal demographic refuge in stochastic environments, but empirical evidence is still lacking of its consequences on temporal population genetic structures. In this long-term study, we investigated how a particular pattern of prolonged diapause may influence the temporal population genetics of the invasive seed-specialized wasp Megastigmus schimitscheki (Hymenoptera: Torymidae) in southeastern France. We characterized the diapause strategy of M. schimitscheki using records of emergence from diapause in 97 larval cohorts, and we conducted a temporal population genetic study on a natural invasive wasp population sampled during ten consecutive years (1999–2008) using polymorphic microsatellite markers. We found that M. schimitscheki can undergo a prolonged diapause of up to five years and displays two main adult emergence peaks after two and four years of diapause. Such a bimodal and atypical pattern did not disrupt temporal gene flow between cohorts produced in even and in odd years during the period of the study. Unexpectedly, we found that this wasp population consisted of two distinct genetic sub-populations that strongly diverged in their diapause strategies, with very few admixed individuals. One of the sub-populations displayed both short and prolonged diapause (2 and 4 years respectively) in equal proportions, whereas the other sub-population displayed mainly short diapause. This study provided empirical evidence that prolonged diapause phenotypes can substantially contribute to reproduction and impact temporal genetic structures. Prolonged diapause is likely to act as both demographic and genetic refuges for insect populations living in fluctuating environments.