Understanding the effect of human-induced landscape fragmentation on gene flow and evolutionary potential of wild populations has become a major concern. Here, we investigated the effect of riverscape fragmentation on patterns of genetic diversity in the freshwater resident European brook lamprey (Lampetra planeri) that has a low ability to pass obstacles to migration. We tested the hypotheses of (i) asymmetric gene flow following water current and (ii) an effect of gene flow with the closely related anadromous river lamprey (L. fluviatilis) ecotype on L. planeri genetic diversity. We genotyped 2472 individuals, including 225 L. fluviatilis, sampled from 81 sites upstream and downstream barriers to migration, in 29 western European rivers. Linear modelling revealed a strong positive relationship between genetic diversity and the distance from the river source, consistent with expected patterns of decreased gene flow into upstream populations. However, the presence of anthropogenic barriers had a moderate effect on spatial genetic structure. Accordingly, we found evidence for downstream-directed gene flow, supporting the hypothesis that barriers do not limit dispersal mediated by water flow. Downstream L. planeri populations in sympatry with L. fluviatilis displayed consistently higher genetic diversity. We conclude that genetic drift and slight downstream gene flow drive the genetic make-up of upstream L. planeri populations whereas gene flow between ecotypes maintains higher levels of genetic diversity in L. planeri populations sympatric with L. fluviatilis. We discuss the implications of these results for the design of conservation strategies of lamprey, and other freshwater organisms with several ecotypes, in fragmented dendritic river networks.