This work presents a multiscale analysis of the fish diversity of the lower St. Lawrence River which flows from the Laurentian Great Lakes of North America. A database of about 14,000 fish sampling stations from the lower St. Lawrence is linked to five different habitat maps of the study area: hydrographical units, water masses, depth, sediment type and wetland type. We hypothesize that species turnover (beta diversity) will be significant among these habitat maps. For each map, we calculate a UPGMA dendrogram based on the Jaccard coefficient of similarity in species occurrences as a first assemblage–habitat model. A randomization test is then used to identify the significant dendrograms from which we infer fish assemblages. We then show that many species are actually selecting or avoiding habitats. Finally, species selecting particular habitats are described by some selected ecological traits that are expected to occur frequently in those habitats. We found assemblages for the hydrographical units but not for the water masses. The fluvial section is selected by many stenohaline and some euryhaline species, while the freshwater estuary and the brackish estuary are only selected by euryhaline species. In the fluvial section at the hydrographical unit scale, many species associated with lentic habitats are limnophilic and more vegetation-dependent while many species selecting lotic habitats are rheophilic insectivorous species. Significant assemblages are defined for depth, sediment type and wetland type. Taken together, we found strong empirical evidence of a diverse actively selecting littoral assemblage of small, low mobility fishes opposing a channel assemblage of larger, more mobile fishes.