Landscapes in Iowa and other midwestern states have been profoundly altered by conversion of native prairies to agriculture. We analyzed landscape data collected at multiple spatial scales to explore relationships with reach-scale physical habitat and fish assemblage data from 93 randomly selected sites on second- through fifth-order wadeable Iowa streams. Ordination of sites by physical habitat showed significant gradients of channel shape, habitat complexity, substrate composition, and stream size. Several landscape variables were significantly associated with the physical habitat ordination. Row crop land use was associated with fine substrates and steep bank angles, whereas wetland land cover and greater sinuosity and catchment land area were associated with complex channel and bank morphology and greater residual pool volume, woody debris, and canopy cover. Thirteen landscape variables were significant predictors of physical habitat variables in multiple linear regressions, with adjusted R ² values ranging from 0.07 to 0.74. Inclusion of landscape variables with physical habitat variables in multiple regression models predicting fish assemblage metrics and a fish index of biotic integrity resulted in negligible improvements over models based on only physical habitat variables. Physical habitat in wadeable Iowa streams is strongly associated with landscape characteristics. Results of this study and previous studies suggest that (1) landscape factors directly influence physical habitat, (2) physical habitat directly influences fish assemblages, and (3) the influence of landscape factors on fish assemblages is primarily indirect. Understanding how landscape factors, such as human land use, influence physical habitat and fish assemblages will help managers make more informed decisions for improving Iowa's wadeable streams.