Abstract Non-native fishes are often blamed for exerting adverse ecological impacts on native fishes, but the evidence is circumstantial and the true impacts remain largely unknown. This dilemma is particularly acute for freshwater ecosystems of the Mediterranean Region, where a high level of endemism (a biodiversity hotspot) makes native fish extinctions much more likely because of their small natural ranges. The aim of this paper is to review non-native fish impacts, from genes to ecosystem level, in the fresh waters of the Mediterranean Region and identify gaps in knowledge that currently hamper effective conservation and management of native species and ecosystems. Most studies reviewed reported potential impacts rather than realised impact mechanisms, and these were conducted predominantly in the European part of the Mediterranean Region. No studies of realised impacts were found for three of the 15 most introduced fishes, which emphasises the need for evidence-based studies of non-native species impacts.