There is considerable uncertainty about the relative roles of stream habitat and landscape characteristics in structuring stream-fish assemblages. We evaluated the relative importance of environmental characteristics on fish occupancy at the local and landscape scales within the upper Little Tennessee River basin of Georgia and North Carolina. Fishes were sampled using a quadrat sample design at 525 channel units within 48 study reaches during two consecutive years. We evaluated species–habitat relationships (local and landscape factors) by developing hierarchical, multispecies occupancy models. Modeling results suggested that fish occupancy within the Little Tennessee River basin was primarily influenced by stream topology and topography, urban land coverage, and channel unit types. Landscape scale factors (e.g., urban land coverage and elevation) largely controlled the fish assemblage structure at a stream-reach level, and local-scale factors (i.e., channel unit types) influenced fish distribution within stream reaches. Our study demonstrates the utility of a multi-scaled approach and the need to account for hierarchy and the interscale interactions of factors influencing assemblage structure prior to monitoring fish assemblages, developing biological management plans, or allocating management resources throughout a stream system.