Habitat fragmentation affects the integrity of many species, but little is known about species-specific sensitivity to fragmentation. Here, we compared the genetic structure of four freshwater fish species differing in their body size (Leuciscus cephalus; Leuciscus leuciscus; Gobio gobio and Phoxinus phoxinus) between a fragmented and a continuous landscape. We tested if, overall, fragmentation affected the genetic structure of these fish species, and if these species differed in their sensitivity to fragmentation. Fragmentation negatively affected the genetic structure of these species. Indeed, irrespective of the species identity, allelic richness and heterozygosity were lower, and population divergence was higher in the fragmented than in the continuous landscape. This response to fragmentation was highly species-specific, with the smallest fish species (P. phoxinus) being slightly affected by fragmentation. On the contrary, fish species of intermediate body size (L. leuciscus and G. gobio) were highly affected, whereas the largest fish species (L. cephalus) was intermediately affected by fragmentation. We discuss the relative role of dispersal ability and effective population size on the responses to fragmentation we report here. The weirs studied here are of considerable historical importance. We therefore conclude that restoration programmes will need to consider both this societal context and the biological characteristics of the species sharing this ecosystem