Summary Genetic divergence between species depends on reproductive isolation (RI) due to traits that reduce interspecific mating (prezygotic isolation) or are due to reduced hybrid fitness (postzygotic isolation). Previous research found that prezygotic barriers tend to be stronger than postzygotic barriers, but most studies are based on the evaluation of F₁ hybrid fitness in early life cycle stages. We combined field and experimental data to determine the strength of 17 prezygotic and postzygotic reproductive barriers between two Lysimachia species that often co‐occur and share pollinators. We assessed postzygotic barriers up to F₂ hybrids and backcrosses. The two species showed near complete RI due to the cumulative effect of multiple barriers, with an uneven and asymmetric contribution to isolation. In allopatry, prezygotic barriers contributed more to reduce gene flow than postzygotic barriers, but their contributions were more similar in sympatry. The strength of postzygotic RI was up to three times lower for F₁ progeny than for F₂ or backcrossed progenies, and RI was only complete when late F₁ stages and either F₂ or backcrosses were accounted for. Our results thus suggest that the relative strength of postzygotic RI may be underestimated when its effects on late stages of the life cycle are disregarded.