For many species, the Mediterranean region harbors distinct lineages that are of conservation concerns. However, many of these are threatened by habitat degradation and by the introduction of non-native species. Here, we assess the status of the native threespine stickleback (Gasterosteus aculeatus) in the Lake Bracciano region in Italy, where stickleback have been historically present. During a dedicated sampling campaign in summer 2015, surveying the potential habitats that sticklebacks commonly occupy, we could not confirm the presence of this species but found introduced species to be often most abundant. Stickleback are thus likely to either have become extinct over the last decades or be on the verge to extinction in the Lake Bracciano region.