Theory predicts that the distribution of genetic diversity in a landscape is strongly dependent on the connectivity of the metapopulation and the dispersal of individuals between patches. However, the influence of explicit spatial configurations such as dendritic landscapes on the genetic diversity and structure of metapopulations is still understudied, and theoretical corroborations of empirical patterns are largely lacking. Here, we used real-world microsatellite data and stochastic simulations of two metapopulations of freshwater amphipods in a 28,000 km2 riverine network to study the influence of spatial connectivity and dispersal strategies on their spatial genetic diversity and structure. We found a significant imprint of the riverine network connectivity on the genetic diversity of both amphipod species. Data from 95 sites showed that allelic richness and observed heterozygosity significantly increased towards more central nodes of the network. In simulation models, dispersal rate was suggested to be the key factor explaining the empirically observed distribution of genetic diversity. Contrary to often-claimed expectations, however, the relevance of directionality of dispersal was only minor. Surprisingly, also the consideration of site-specific carrying capacities, for example by assuming a direct dependency of population size with local river size, substantially decreased the model fit to empirical data. This highlights that directional dispersal and the spatial arrangement of population sizes may have a smaller relevance in shaping population genetic diversity of riverine organisms than previously thought, and that dispersal along the river network is the single-most important determinant of population genetic diversity.