Understanding the broad-scale factors that influence biological communities has long been a goal of community ecology. We used benthic macroinvertebrate data to identify broad geographical patterns in macroinvertebrate community composition and specifically to examine the influence of the Precambrian Shield on stream abiotic and biotic conditions. The Precambrian Shield is a geological feature that encompasses most of northern North America. Geology differs between Shield and off-Shield areas, creating distinctly different physical and chemical conditions in aquatic systems. We focused our regional-scale study on south-central Ontario, where both Shield and off-Shield conditions are found in adjacent areas. We used constrained and unconstrained multivariate analyses to examine patterns in biotic, abiotic, and spatial variables. Our results showed that, in low-order lotic systems, macroinvertebrate communities differ between Shield and off-Shield streams. Shield streams have higher dissolved O₂, velocity, and discharge, larger amounts of woody debris, and greater canopy cover than off-Shield streams. In contrast, off-Shield streams have higher conductivity, alkalinity, pH, turbidity, and water temperature, and frequently are surrounded by meadow, cultivated, or pastured land. In general, macroinvertebrate communities at off-Shield sites had a greater proportion of taxa preferring pool or depositional habitats, whereas macroinvertebrate communities at Shield sites contained taxa typically associated with riffles or erosional habitats. Analysis of spatial location indicated that the Shield/off-Shield distinction probably is the result of a combination of intertwined abiotic and spatial factors.