This study evaluated the effects of spatial scale and spatial extent on the factors governing fish assemblage patterns in southern Ontario streams. Multivariate analyses were used to partition the variation in environmental factors among three scales (in-stream habitat, watershed and regional). Results indicate that watershed features are the best predictors of assemblage patterns, followed closely by in-stream habitat and regional factors; however, there was considerable interaction among scales. Base-flow index and biogeography proved to be the two most influential factors explaining assemblage patterns across southern Ontario. When spatial extent was reduced to the individual basins, it was evident that watershed features continue to explain more variation than in-stream habitat. Although the primary gradients explaining assemblage patterns varied among the basins, assemblages were arranged in a predictive manner along a base flow-forest cover-water temperature gradient. Results of this study emphasize the importance of regional and landscape conditions on stream-fish assemblages while underscoring the benefits of using a mutli-scaled approach to elucidate the primary gradients across large geographical extents.