Estimating genetic connectivity in disturbed riverine landscapes is of key importance for river restoration. However, few species of the disturbed riverine fauna may provide a detailed and basin-wide picture of the human impact on the population genetics of riverine organisms. Here we used the most abundant native fish, the three-spined stickleback (Gasterosteus aculeatus L.), to detect the geographical determinants of genetic connectivity in the eastern part of the Scheldt basin in Belgium. Anthropogenic structures came out as the strongest determinant of population structure, when evaluated against a geographically well-documented baseline model accounting for natural effects. These barriers not only affected genetic diversity, but they also controlled the balance between gene flow and genetic drift, and therefore may crucially disrupt the population structure of sticklebacks. Landscape models explained a high percentage of variation (allelic richness: adjusted R2 = 0.78; pairwise FST: adjusted R2 = 0.60), and likely apply to other species as well. River restoration and conservation genetics may highly benefit from riverine landscape genetics, including model building, the detection of outlier populations, and a specific test for the geographical factors controlling the balance between gene flow and genetic drift.