One of the longest debates in biology has been over the relative importance of different isolating barriers in speciation. However, for most species, there are few data evaluating their relative contributions and we can only speculate on the general roles of pre- and postzygotic isolation. Here we quantify the absolute and cumulative contribution of 198 potential reproductive barriers between two sympatric damselfly sister species, Ischnura elegans and I. graellsii, including both premating (habitat, temporal, sexual and mechanical isolation) and postmating barriers (prezygotic: sperm insemination success and removal rate, oviposition success, fertility, fecundity; postzygotic: hybrid viability, hybrid sterility and hybrid breakdown). In sympatry, total reproductive isolation between I. elegans females and I. graellsii males was 95.2%, owing mostly to a premating mechanical incompatibility (93.4%), while other barriers were of little importance. Isolation between I. graellsii females and I. elegans males was also nearly complete (95.8%), which was caused by the cumulative action of multiple prezygotic (n=4, 75.4%) and postzygotic postmating barriers (n=5, 7.4%). Our results suggest that premating barriers are key factors in preventing gene flow between species, and that the relative strengths of premating barriers is highly asymmetrical between the reciprocal crosses.