Abstract Negative interspecific mating interactions, known as reproductive interference, can hamper species coexistence in a local patch and promote niche partitioning or geographical segregation of closely related species. Conspecific sperm precedence (CSP), which occurs when females that have mated with both conspecific and heterospecific males preferentially use conspecific sperm for fertilization, might contribute to species coexistence by mitigating the costs of interspecific mating and hybridization. We examined whether two closely related species exhibiting CSP can coexist in a local environment in the presence of reproductive interference. First, using a behaviourally explicit mathematical model, we demonstrated that two species characterized by negative mating interactions are unlikely to coexist because the costs of reproductive interference, such as loss of mating opportunity with conspecific partners, are inevitably incurred when individuals of both species are present. Second, we experimentally demonstrated differences in mating activity and preference in two Harmonia ladybird species known to exhibit CSP. According to the developed mathematical model of reproductive interference, these behavioural differences should lead to local extinction of H. yedoensis because of reproductive interference by H. axyridis . This prediction is consistent with field observations that H. axyridis uses various food sources and habitats whereas H. yedoensis is confined to a less preferred prey item and a pine tree habitat. Finally, by a comparative approach, we showed that niche partitioning or parapatric distribution, but not sympatric coexistence in the same habitat, is maintained between species with CSP belonging to a wide range of taxa, including vertebrates and invertebrates living in aquatic or terrestrial environments. Taken together, these results lead us to conclude that reproductive interference generally destabilizes local coexistence even in closely related species that exhibit CSP.