Livingstone's fruit bat (Pteropus livingstonii) is among the most threatened bats worldwide. As the species is an important pollinator and seed disperser of Comoros plants with a crucial role in forest regeneration, it is critical to understand how human‐induced habitat disturbance impacts genetic structure at fine spatial scales in order to define conservation strategies. In this study, we assessed the level of genetic structure between eight colonies of the species and found that this was strong genetic structures between roosts that can be explained by isolation due both to distance and habitat fragmentation. Mitigation measures should aim to restore population connectivity.