The Rhine catchment in Switzerland has been transformed by a chain of hydroelectric power stations. We addressed the impact of fragmentation on the genetic structure of fish populations by focusing on the European chub (Squalius cephalus). This fish species is not stocked and copes well with altered habitats, enabling an assessment of the effects of fragmentation per se. Using microsatellites, we genotyped 2133 chub from 47 sites within the catchment fragmented by 37 hydroelectric power stations, two weirs and the Rhine Falls. The shallow genetic population structure reflected drainage topology and was affected significantly by barriers to migration. The effect of power stations equipped with fishpasses on genetic differentiation was detectable, albeit weaker than that of man-made barriers without fishpasses. The Rhine Falls as the only long-standing natural obstacle (formed 14 000 to 17 000 years ago) also had a strong effect. Man-made barriers also exacerbated the upstream decrease in allelic diversity in the catchment, particularly when lacking fishpasses. Thus, existing fishpasses do have the desired effect of mitigating fragmentation, but barriers still reduce population connectivity in a fish that traverses fishpasses better than many other species. Less mobile species are likely to be affected more severely.