Gene flow in riverine species is constrained by the dendritic (branching) structure of the river network. Spatial genetic structure (SGS) of freshwater insects is particularly influenced by catchment characteristics and land use in the surroundings of the river. Gene flow also depends on the life cycle of organisms. Aquatic larvae mainly drift downstream whereas flying adults can disperse actively overland and along watercourses. In-stream movements can generate isolation by distance (IBD) at a local scale and differentiation between subcatchments. However, these patterns can be disrupted by overland dispersal. We studied SGS across the Loire River in the damselfly Calopteryx splendens which is able to disperse along and between watercourses. Our sampling design allowed us to test for overland dispersal effects on genetic differentiation between watercourses. Amplified fragment length polymorphism markers revealed high genetic differentiation at the catchment scale but the genetic structure did not reflect the geographical structure of sampling sites. We observed IBD patterns when considering the distance following the watercourse but also the Euclidean distance, i.e. the shortest distance, between pairs of sites. Altogether, our results support the hypothesis of overland dispersal between watercourses. From a conservation perspective, attention should be paid to the actual pathways of gene flow across complex landscapes such as river networks.