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The brook chub (Squalius lucumonis) is a freshwater fish, endemic of Central Italy, which is experiencing a rapid range decline so that it is presently listed as Critically Endangered in the Italian IUCN Red List. For effective conservation, information about the spatial pattern of genetic diversity is crucial. Therefore, we analysed the mitochondrial Control Region and nuclear (microsatellites) markers to investigate population genetic structure, demography and spatial diversity over the whole species distribution range. We revealed significant divergence among populations, even at the local spatial scale, according to the isolation by distance model. At the biogeographic spatial scale, genetic diversity was shaped by past hydrogeological and climatic events that isolated the principal drainage basins (Vara, Tiber and Arno) from each other. On the other hand, strong genetic differentiation within the Tiber drainage basin could be due to local factors that acted at single-stream scale, as recent barriers to fish dispersal and irregular seasonal flow rates typical of small Mediterranean streams. Our findings contribute to the basal data collection on S. lucumonis required by European Habitats Directive and necessary for planning protection actions. We recommend that the three river drainages and most of the sampling sites should be regarded as different Management Units (MUs) to preserve their genetic distinctiveness. A recovery plan for the brook chub should consider environmental intervention and creation of protected areas, as well as in situ/ex situ restocking activities with juveniles produced by breeders from the same MU, to preserve local (adaptive) diversity.