PremiseBarriers at different reproductive stages contribute to reproductive isolation. Self‐incompatibility (SI) systems that prevent self‐pollination could also act to control interspecific pollination and contribute to reproductive isolation, preventing hybridization. Here we evaluated whether SI contributes to reproductive isolation among four co‐occurring Opuntia species that flower at similar times and may hybridize with each other.MethodsWe assessed whether Opuntia cantabrigiensis, O. robusta, O. streptacantha, and O. tomentosa, were self‐compatible and formed hybrid seeds in five manipulation treatments to achieve self‐pollination, intraspecific cross‐pollination, open pollination (control), interspecific crosses or apomixis, then recorded flowering phenology and synchrony.ResultsAll species flowered in the spring with a degree of synchrony, so that two pairs of species were predisposed to interspecific pollination (O. cantabrigiensis with O. robusta, O. streptacantha with O. tomentosa). All species had distinct reproductive systems: Opuntia cantabrigiensis is self‐incompatible and did not produce hybrid seeds as an interspecific pollen recipient; O. robusta is a dioecious species, which formed a low proportion of hybrid seeds; O. streptacantha and O. tomentosa are self‐compatible and produced hybrid seeds.ConclusionsOpuntia cantabrigiensis had a strong pollen–pistil barrier, likely due to its self‐incompatibility. Opuntia robusta, the dioecious species, is an obligate outcrosser and probably partially lost its ability to prevent interspecific pollen germination. Given that the self‐compatible species can set hybrid seeds, we conclude that pollen–pistil interaction and high flowering synchrony represent weak barriers; whether reproductive isolation occurs later in their life cycle (e.g., germination or seedling survival) needs to be determined.