Despite the value of aquatic insects in aquatic ecosystem biomonitoring, few studies within North America have addressed relationships between aquatic insect assemblages and landscape-level environmental variables. In this study, over 300,000 adult caddisfly specimens representing 224 species were collected and analyzed from samples of 225 Minnesota aquatic habitats within 58 watersheds. Detrended Correspondence Analysis and a UPGMA dendrogram of caddisfly relative abundance data determined that five regions of caddisfly biodiversity exist within the state. Species richness and diversity were significantly highest in the Lake Superior and Northern regions, lowest in the Northwestern and Southern regions, and intermediate in the Southeastern region. Canonical Correspondence Analysis determined that caddisfly species composition was related to temperature, percentage of disturbed habitat, and stream gradient. Although a strong correlation between temperature and percentage of disturbed habitat made determination of the relative importance of those variables difficult, it is likely that anthropogenic disturbance has decreased caddisfly biodiversity in at least the Northwestern and Southern regions. Now that regions of biodiversity have been established, future changes to the fauna can be evaluated with greater precision and confidence. This study represents the most comprehensive faunal analysis of an aquatic insect order within the Western Hemisphere.