AbstractIn this study, we investigated functional structure patterns of tropical headwater and river fish assemblages. We hypothesised that environmental conditions are primarily structuring headwater streams leading to functionally clustered assemblages, whereas processes that favour functional overdispersion would guide river assemblages. For 27 headwater streams and 22 rivers, we used eight functional traits for calculating two functional indexes: mean pairwise distance (MPD) and net relatedness index (NRI). We performed linear regressions between indexes and species richness, a multiple regression between NRI and eight environmental variables and a variation partitioning to disentangle the role of environment and space on NRI. Our findings indicate that fish assemblages of headwaters are structured by environmental conditions as most assemblages in this habitat displayed a tendency to clustering and MPD/NRI were not correlated with species diversity, whereas the opposite pattern was observed for river habitat. Four environmental variables (channel depth, water velocity, dissolved oxygen and turbidity) explain 56% of functional structure variation. These variables seem to function as selective filters in headwaters, whereas channel depth may be determinant for functional overdispersion of river fish assemblages. Components associated with space are also influencing the functional structure. Limitations of species dispersal through space (between both habitat types) appear as a possible cause to this. In this sense, both environmental conditions and processes linked with space are capable of influencing the functional structure of tropical headwater streams and river fish assemblages.