Traditionally, species richness and composition of freshwater communities are expected to change according to the structural variation from headwaters to river mouth. In the present study, we hypothesised that fish functional traits will respond to this gradient, leading to functional dissimilarities between fish assemblages of headwaters and those of rivers. We addressed the following questions: (1) which functional traits are present in fish assemblages of each habitat type; (2) which environmental factors determine the occurrence of fish functional traits; and (3) how functional traits are related to each environmental variable? Our findings showed dissimilarities between fish assemblages of headwaters and those of rivers, mainly driven by channel depth, channel width, water temperature, water turbidity and concentration of dissolved oxygen, whereas altitude, water temperature and pH were mainly correlated with variation within each habitat type. These results suggested that not only do the physical variables proposed in classical theory of the river-continuum concept follow the upstream–downstream gradient in structuring the functional organisation of fish assemblages, but that also physicochemical variables, such as turbidity and concentration of dissolved oxygen, display this trend in tropical river systems. Moreover, our results have given a first perspective on what kind of fish species and fish functional traits to expect in each habitat type and, consequently, along the headwater–river mouth gradient.