River-floodplain systems host very high biodiversity and provide a great variety of ecosystem services due largely to their high environmental heterogeneity, which is strongly dependent on spatial and temporal scales of observation. We aimed to describe the main patterns of environmental variation of the subtropical system of the Middle Paraná River at different scales of analysis using univariate and multivariate statistics. Physical, chemical and biological variables were analyzed during different hydro-sedimentological phases along four consecutive years in 31 aquatic environments. We considered four aquatic habitat types with different degrees of hydrological connectivity and water flow characteristics (major rivers, secondary channels, floodplain lakes permanently connected to the major rivers, and floodplain lakes temporarily connected to the major rivers) from two geomorphologic units with different drainage patterns: anastomosing and meandering. The environmental heterogeneity was mainly associated with the habitat types and relative contributions of lateral tributaries with different water qualities to each unit, in combination with the influence of the hydro-sedimentological regime. The heterogeneity of the system was significantly higher during low water than during high water phases. However, although the extreme flood reduced dissimilarities between habitat types within each geomorphologic unit, it increased differences between major rivers and between units. In conclusion, at different spatial scales, floods may simultaneously have a homogenization effect due to increased hydrological connectivity and a differentiation effect due to enhanced inputs of water from regions with variable geological characteristics and land uses. Finally, geomorphologic units can play a key role in maintaining the environmental heterogeneity during floods of high magnitude, even when land barriers among aquatic environments disappear.