Aim: The aim of this study was to determine whether the Casiquiare River functions as a free dispersal corridor or as a partial barrier (i.e. filter) for the interchange of fish species of the Orinoco and Negro/Amazon basins using species assemblage patterns according to geographical location and environmental features. Location: The Casiquiare, Upper Orinoco and Upper Negro rivers in southern Venezuela, South America. Methods: Our study was based on an analysis of species presence/absence data and environmental information (11 habitat characteristics) collected by the authors and colleagues between the years 1984 and 1999. The data set consisted of 269 sampled sites and 452 fish species (> 50,000 specimens). A wide range of habitat types was included in the samples, and the collection sites were located at various points along the entire length of the Casiquiare main channel, at multiple sites on its tributary streams, as well as at various nearby sites outside the Casiquiare drainage, within the Upper Orinoco and Upper Rio Negro river systems. Most specimens and field data used in this analysis are archived in the Museo de Ciencias Naturales in Guanare, Venezuela. We performed canonical correspondence analysis (CCA) based on species presence/absence using two versions of the data set: one that eliminated sites having < 5 species and species occurring at < 5 sites; and another that eliminated sites having < 10 species and species occurring at < 10 sites. Cluster analysis was performed on sites based on species assemblage similarity, and a separate analysis was performed on species based on CCA loadings. Results: The CCA results for the two versions of the data set were qualitatively the same. The dominant environmental axis contrasted assemblages and sites associated with blackwater vs. clearwater conditions. Longitudinal position on the Casiquiare River was correlated (r2 = 0.33) with CCA axis-1 scores, reflecting clearwater conditions nearer to its origin (bifurcation of the Orinoco) and blackwater conditions nearer to its mouth (junction with the Rio Negro). The second CCA axis was most strongly associated with habitat size and structural complexity. Species associations derived from the unweighted pair-group average clustering method and pair-wise squared Euclidean distances calculated from species loadings on CCA axes 1 and 2 showed seven ecological groupings. Cluster analysis of species assemblages according to watershed revealed a stronger influence of local environmental conditions than of geographical proximity. Main conclusions: Fish assemblage composition is more consistently associated with local environmental conditions than with geographical position within the river drainages. Nonetheless, the results support the hypothesis that the mainstem Casiquiare represents a hydrochemical gradient between clearwaters at its origin and blackwaters at its mouth, and as such appears to function as a semi-permeable barrier (environmental filter) to dispersal and faunal exchanges between the partially vicariant fish faunas of the Upper Orinoco and Upper Negro rivers. ?? 2008 The Authors.