Transgenic crops expressing Cry proteins are widely cultivated to reduce crop damage from insect pests. However, Cry proteins can leach from transgenic crop detritus into nearby aquatic ecosystems, potentially affecting non-target organisms. Here, we assess the impacts of exposure to a commonly used Cry protein, Cry1Ab, on net-spinning caddisflies (Hydropsychidae), a common and ecologically important group of stream insects. We collected and genetically barcoded 1862 larval caddisflies from 25 streams across the Midwestern United States, comparing populations from Cry1Ab-positive and Cry1Ab-negative streams. We evaluated potential impacts associated with body size, developmental stage, species assemblages, and mitochondrial haplotype diversity. Overall, we observed limited effects of Cry1Ab exposure, including most comparisons showing no difference between Cry1Ab-positive and Cry1Ab-negative streams with a few variable exceptions. Second instar Hydropsyche betteni showed significantly reduced body size at Cry1Ab-positive sites, while two other species showed size increases. Mean developmental stage within species and species assemblages showed no consistent association with Cry1Ab. In contrast, environmental variables such as water temperature, stream width, and watershed land use were stronger predictors of body size, instar progression, and species assemblages. Haplotype networks revealed strong geographic structure but no genetic patterns related to Cry1Ab exposure. These results indicate that Bt-Cry exposure is not a major driver of morphological, developmental, or genetic variation in Hydropsychid caddisflies under natural field conditions. Environmental gradients remain the dominant structuring force, although further work is needed to assess potential context-dependent or cumulative effects of transgenic crop byproducts in aquatic ecosystems.