Aim Dendritic ecological networks (DENs), such as river systems, combine features that challenge the traditional conceptual views and empirical approaches applied to metacommunities. As a result of their dendritic branching geometry and stream flow directionality, they are strongly hierarchical and asymmetrical. We analysed the metacommunity structure of benthic diatoms in a large-scale river system with the aim of evaluating the importance of potential causal influences. Furthermore, we hypothesized that metacommunities of diatoms that are strongly attached to their substrata show a different spatial structure than metacommunities of other, more weakly attached diatoms. Location The study was carried out in the Dong River, a 32,275 km2 subtropical river network located in southern China. Methods We surveyed benthic diatom communities during three seasons (dry, intermediate and wet). Using partial redundancy analysis, we partitioned community variation among environmental models and different spatial eigenfunction models to evaluate the influence of alternative dispersal pathways (overland versus water course dispersal), stream directionality, man-made dams and diatom functional traits on diatom metacommunity structure. Results Models based on hydrological connections and water directionality represent spatial patterns better than overland distances, suggesting that the dynamics of benthic diatom metacommunities are mainly confined to the river network and influenced by the prevailing water flow. We found significant effects of man-made dams on the spatial structure of important limnological variables and diatom metacommunity structure. The metacommunity of strongly attached diatoms also showed a weaker signature of flow directionality than that of other growth forms, especially in seasons with high water levels. Main conclusions We conclude that the consideration of among-site connectivity, flow directionality and species traits is key to a better understanding of the spatial ecology of passively dispersing microbial organisms in river systems.