Our lack of understanding of relationships between stream biotic communities and surrounding landscape conditions makes it difficult to determine the spatial scale at which management practices are best assessed. We investigated these relationships in the Minnesota River Basin, which is divided into major watersheds and agroecoregions which are based on soil type, geologic parent material, landscape slope steepness, and climatic factors affecting crop productivity. We collected macroinvertebrate and stream habitat data from 68 tributaries among three major watersheds and two agroecoregions. We tested the effectiveness of the two landscape classification systems (i.e., watershed, agroecoregion) in explaining variance in habitat and macroinvertebrate metrics, and analyzed the relative influence on macroinvertebrates of local habitat versus regional characteristics. Macroinvertebrate community composition was most strongly influenced by local habitat; the variance in habitat conditions was best explained at the scale of intersection of major watershed and agroecoregion (i.e., stream habitat conditions were most homogeneous within the physical regions of intersection of these two landscape classification systems). Our results are consistent with findings of other authors that most variation in macroinvertebrate community data from large agricultural catchments is attributable to local physical conditions. Our results are the first to test the hypothesis and demonstrate that the scale of intersection best explains these variances. The results suggest that management practices adjusted for both watershed and ecoregion characteristics, with the goal of improving physical habitat characteristics of local streams, may lead to better basin-wide water quality conditions and stream biological integrity.