© 2021 Elsevier B.V.Seasonal and annual dynamics of the zooplankton community in lakes are affected by changes in abiotic drivers, trophic interactions (e.g., changes in phytoplankton and fish communities and abundances) and habitat characteristics (e.g. macrophyte abundance and composition). However, little is known about the temporal responses of the zooplankton community to abiotic and biotic drivers across lakes at the regional scale. Using a comprehensive 20-year dataset from 20 Danish lakes in recovery from eutrophication, we assessed the seasonal and annual trends in the spatial heterogeneity of zooplankton community across lakes and related it to abiotic and biotic drivers. We found significant seasonality and inter-annual decreases in spatial zooplankton heterogeneity in both shallow and deep lakes, with the decrease in the spatial turnover dominating the temporal dynamics of the beta diversity. For the inter-annual changes, decreased spatial heterogeneity of phytoplankton, macrophytes and fish were important biotic drivers at the regional scale. Using a series of ordinary least squares regressions and model selection with model averaging approaches, we revealed that both local (e.g., total phosphorus, total nitrogen, pH, Secchi depth, alkalinity, Schmidt stability, water temperature) and regional drivers (e.g., air temperature, solar irradiance) were important variables influencing the spatial zooplankton heterogeneity, although the directions depended on the beta diversity measures and water depth. Our results highlight an important role of bottom-up forces through phytoplankton community as well as macrophytes and top-down forces via fishes in driving the temporal changes in zooplankton community composition patterns at the regional scale.