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We applied a multiscale approach to understand the patterns of longitudinal, lateral, and temporal variability in fish assemblage structure of a large floodplain river and explored their relationships with climatic seasonality, hydrological variability, and floodplain geomorphology. We studied the fish assemblage structure of 35 connected lakes along a 600-km section of the Paraná River over 5 y. Longitudinal changes in fish assemblage structure were associated with a latitudinal climatic gradient, whereas temporal changes were associated with climatic seasonality and hydrological variability. Longitudinal and seasonal changes in assemblage structure were probably based on different thermal preferences of species that migrate seasonally along the main river axis. Extraordinary floods in summer promoted downstream dispersal of several species, which increased similarity between downstream and upstream regions. Thermal seasonality of this subtropical system was a strong factor driving temporal changes in assemblage structure, and the effects of the hydrological regime largely depended on flood pulses coincident with periods of warmer temperatures. Fish assemblages varied secondarily along a lateral gradient represented by types of geomorphic units distributed in the floodplain. The geomorphic units generated patchy environmental heterogeneity at the landscape scale that resulted in discontinuous patterns of fish assemblage structure. Assemblage composition was more similar among lakes belonging to similar geomorphic units but situated at geographically distant regions than among lakes belonging to different geomorphic units situated in the same region. Therefore, patterns in fish assemblage variation at regional and landscape scales appear to be determined largely by the dispersal and selection of suitable habitats by fish across the hierarchy of spatial and temporal scales. [ABSTRACT FROM AUTHOR]