Fish assemblages play a key role in stream ecosystems, and play a crucial role in the assessment of stream health. Physical habitat is a key component of stream ecosystems and plays a major role in determining biotic assemblages and stream integrity. There is increasing recognition of the role landscape factors play in determining biotic assemblages and stream integrity. Landscapes in Iowa and other Midwestern states have been profoundly altered by conversion of native prairies and wetlands to agriculture. We analyzed fish assemblages, physical habitat and landscape characteristics from 93 randomly selected sites on wadeable Iowa streams to explore relationships between fish assemblage, reach scale physical habitat and landscape characteristics at multiple spatial scales. Non-metric multidimensional scaling ordination and stepwise multiple regression were used to explore, identify and quantify relationships. Ordination of sites by species abundance showed significant gradients related to stream size and stream health. Thirty variables were identified as significantly correlated to the ordination of fish assemblage and significantly differed between healthy and impaired sites. Eighteen variables of physical habitat were identified as predictors of fish assemblage metrics. Variables described channel morphology, channel cross section and bank morphology, residual pool volume, relative bed stability, large woody debris, riparian vegetation, fish cover, human disturbance and substrate composition. Fish assemblages in Iowa wadeable streams are associated with the quality of the instream physical conditions that constitute an important part of their habitat. Ordination of sites by physical habitat showed significant gradients of channel shape and habitat complexity, substrate composition and stream size. Land cover variables were strongly correlated with channel shape and habitat complexity, and catchment land area and gradient were correlated with stream size. Fish assemblage was associated with gradients of land cover and stream size. Our results support hierarchical stream system theory and support the view that landscape factors strongly influence mainly physical habitat characteristics in streams, and that in turn these physical habitat characteristics strongly influence stream fish assemblages.