Much is known about invertebrate community traits in basins across Europe, but no comprehensive description of traits exists for the continental US. Little is known about the trait composition of invertebrates in reference or least-disturbed basins of the US, how trait composition varies among ecoregions, or how consistently traits respond to land use. These elements are essential to development of trait-based tools for conservation and assessment of biological integrity. We compared invertebrate traits of least-disturbed basins among ecoregions of the US. Benthic invertebrate data (presence/absence) from 1987 basins were translated into 56 binary traits (e.g., bivoltine, clinger). Basins were classified as least-disturbed, agricultural, or urban, and grouped into 9 ecoregions. Landuse, climatic, physiographic, and hydrologic data were used to describe ecoregions and to evaluate least-disturbed basin quality. The unique habitat template of each ecoregion selected for trait compositions in least-disturbed basins that differed among ecoregions. Among the traits examined, life-history (e.g., voltinism, development) and ecological traits (e.g., rheophily, thermal preference) differed most among ecoregions. Agricultural and urban land uses selected for trait compositions that differed from least-disturbed, but the extent of the differences depended on ecoregion and quality of the least-disturbed basins. No trait compositions unique to specific land uses were found. However, a disturbance syndrome was observed in that the magnitude and direction of trait responses to urban and agricultural land uses were consistent among ecoregions. Each ecoregion had a unique trait composition, but trait compositions could be used to aggregate ecoregions into 3 broad regions: Western Mountains, Plains and Lowlands, and Eastern Highlands. Our results indicate that large-scale trait-based assessment tools for the US will require calibration to account for regional differences in the trait composition of basins and in the quality of least-disturbed basins.