A multi-faceted approach is needed to better understand how the beta diversity of aquatic assemblages responds to ecological gradients. Using distance-based RDA and variance partitioning, we explored the different components of total beta diversity (replacement and richness difference) based on taxonomic, functional, and phylogenetic data of aquatic macrophytes along environmental and spatial gradients across 49 tropical floodplain lakes in the Pantanal of Mato Grosso in the dry season. We found that taxonomic beta diversity was driven by species replacement, while functional beta diversity was mostly contributed by richness difference and phylogenetic beta diversity was equally contributed by both components. Taxonomic and functional beta diversities were driven by environmental variables, connectivity variables, and directional spatial processes. Phylogenetic beta diversity was mainly affected by the connectivity between the sites and their distance from the river. The relationship between environmental variables and beta diversity indicates that a selection of species with similar functional and phylogenetic characteristics co-occurs at a site. The low connectivity between sites and the increased distance between the lakes and the river suggested dispersal limitation, which was due to the absence of directional exchange of species between lakes. Our findings provided new insights into the beta diversity patterns of aquatic macrophytes and their underlying causes in tropical floodplains.