Channel networks of floodplain rivers are the main routes for water, sediment, organic matter, and aquatic organisms between main channels and lakes, playing a substantial role in floodplain dynamics. Despite their importance most investigations in large floodplain rivers focus on main channels and lentic water bodies. This research deals with fish assemblage variations in different lotic sites (scour holes and straight reaches) of one of the many channels which shape the wide anabranching floodplain of the middle reach of the Paraná River. We considered the influence of environmental factors, such as hydro-geomorphological features and water quality of the channel, connections with surrounding lentic water bodies, seasonality, and interannual flow variations. The results showed two main ecological patterns closely related to floodplain connectivity. In-channel habitat heterogeneity has low influence on fish assemblages when minor channels are connected to significant flooded areas (sampling period 2013-2016). Spatial or temporal predictions of fish structure during these stages are not easy due to the interaction of several environmental factors. However, during very low water levels (winter of 2018) most lentic areas become isolated from the nearby channel network, and floodplain fish are forced to move into the numerous scour holes of the floodplain channels where water conditions resemble those in lentic habitats. Our work highlights the importance of in-channel characteristics of floodplain river networks and their crucial role for fish conservation, particularly during very low water stages.