Similarity between assemblages can be explained by environmental and geographic distances, which are directly linked to environmental filtering and dispersal capacity of the species, respectively. Floodplains are typically heterogeneous, creating a situation where environmentally similar habitat patches are more distant geographically than environmentally different ones, what aids the understanding of the importance of each distance to assemblage similarity. Furthermore, flow type and macrophyte presence are important habitat features that influence both environmental conditions and dispersion of species. Thus, we aimed to investigate the influence of these distances on fish assemblage similarity in a Neotropical floodplain, controlling for macrophyte presence and habitat flow. We tested our predictions using three rivers in Upper Paraná River floodplain (Baía, Ivinhema and Paraná rivers). First, we tested the difference in fish assemblage similarity considering the three factors (flow, macrophyte presence and river) with a PERMANOVA on presence-absence data. We then correlated assemblage similarity (indexed by Jaccard similarity) to environmental distance (indexed by Gower dissimilarity) and geographic distances between sites (in km). Regarding the importance of the environmental heterogeneity, the presence of the macrophytes in lentic environments changed significantly the fish fauna while in semilotic environments, assemblages in sites with and without macrophytes were similar. Both geographic and environmental distances were correlated with similarity in lentic sites and in the absence of macrophytes. With macrophytes and in semilotic environments, the effects of these distances were not significant, because these factors act like environmental filters to pre-adapted species, independently of the dispersal distance.