AbstractThe spatial distribution of biological communities may be regulated by environmental and spatial processes, and the intensity of these processes depend on the species characteristics, spatial heterogeneity and spatial extent. We investigated the relationship between the non‐migratory fish metacommunity with environmental and spatial variables using the variance partitioning procedure. We used data from lakes of four Neotropical floodplains: Amazon, Araguaia, Pantanal and Paraná. We hypothesised that the processes that drive the spatial distribution of non‐migratory fish in floodplain systems depend on connectivity driven by flood pulse. We predict that spatial variables are less important to explaining the fish community structures during rainy periods when there is no dispersal limitation. In contrast, environmental variables influence these structures during rainy and dry periods. During dry periods, metacommunities depend on species' dispersal abilities, but during rainy and dry periods, spatial distribution depends on the environmental filters selected by each species. For most of the environmental variables, it was possible to observe more similarity in the rainy period. Our results showed different patterns of fish communities for the lakes of the four floodplains systems, which led us to reject our hypothesis. Non‐migratory fish remain in their natal environment and perform only small lateral movements. In this scenario, we found a little influence from spatial variables in both periods and a great contribution of environmental variables for Amazon (16%) and Pantanal (32%) floodplains in structuring fishes' distribution in the rainy period.