Mountain building reorganizes drainage networks, influencing riverine biodiversity. Northern Italy offers a natural experiment in the impact of tectonic and geomorphic processes on aquatic species distributions. We combined geomorphic analysis with environmental DNA from rivers to assess the influence of tectonically driven drainage reorganization on genetic diversity, targeting an endemic fish species, Telestes muticellus (A. Risso, 1827). In the Northern Apennines, horizontal shortening and topographic advection in an orogenic wedge have been hypothesized as leading to river capture and drainage divide migration. In addition, slab rollback has produced a spatial transition from contraction to extension, which is more pronounced from north to south, with normal faulting producing range-parallel drainage only in the southern regions. In contrast, the adjacent Ligurian Alps are a remnant of the Alpine orogen with little modern deformation. We found distinct zones of geomorphic characteristics from north to south, including divide asymmetry and frequency of range-parallel drainage. Analysis of DNA sequences shows cross-divide assemblage characteristics that correlate with the geomorphic zonation. The Northern Apennines show higher values of the directional measures of assemblage change gain, loss, and turnover; the Ligurian Alps show higher values of overlap and nestedness. There is a positive correlation between divide asymmetry and genetic distance, and gain, loss, and turnover of DNA sequences from Adriatic to Ligurian sites; there is a negative correlation with overlap and nestedness. Since the species is confined to freshwater environments, tectonically driven drainage reorganization is one of the only mechanisms that can explain its spatial genetic differentiation.