Dam construction is widespread, changing the hydrological and biogeochemical conditions and thereby the bacterial communities in the rivers of the earth. To date, knowledge is lacking about bacterial communities in cascade reservoirs. Here, we investigated the bacterial communities and potential functions of nine cascade hydropower reservoirs in 1290 km of the upper Mekong River (Lancang River in China). Along the reservoir cascade, the water temperature, rather than the presence of dams, was the main cause for the geographical patterns of bacterial community composition. Within a reservoir, significant spatial differences in sediment bacterial communities were observed between the tail, middle, and head of a reservoir. The differences in sediment properties resulted by flow velocity-sieved sedimentation from the tail to head of a reservoir caused the spatial variation in sediment bacteria communities, forming potential hotspots for biogeochemical cycling in the middle of the reservoir. In contrast, unlike in deep lakes and deep single reservoirs, the bacterioplankton community composition had no distinctly layered features in the deep cascade reservoirs, because density-induced underwater currents and convection resulting from hydropower production reduced the vertical hydro-environmental gradients. This study provides a novel perspective on the processes affecting the distribution and function of bacterial communities in river-reservoir cascades, and is a first step toward forecasting the consequences of microbially mediated biogeochemical cycling in existing and future reservoirs worldwide.