A functional traits approach was adopted to examine patterns of fish diversity in relation to environmental and spatial variables and for comparison with findings from earlier analyses of fish taxonomic diversity in a nearly pristine river in the Llanos region of Colombia. Fishes were surveyed during the low-water period at 34 sites along the longitudinal fluvial gradient of the Bita River, a clearwater tributary of the Orinoco River. We compiled a matrix of 37 traits associated with locomotion, feeding, and defense for 132 species to compute functional β diversity metrics among four reaches along the entire longitudinal fluvial gradient and also within reaches. Functional redundancy (trait under-dispersion) was found to be common throughout the fluvial gradient. Functional β diversity was high at both spatial scales, with widespread evidence of functional nestedness and functional turnover, especially in upper reaches. In the Bita River, environmental filtering and stochastic processes, such as random dispersal, appear to be the dominant mechanisms influencing fish functional diversity. Inferences derived from analysis of functional versus taxonomic diversity are largely congruent, although analysis of functional β diversity found more evidence of nestedness than turnover at both spatial scales. Both lines of evidence stress the importance of habitat heterogeneity and aquatic habitat connectivity for conservation of high species diversity in this system.