ABSTRACTWhether it is for water supply, flood control or hydropower uses, the transformation of a river into a reservoir can impact freshwater ecosystems and their biodiversity. Using the National Lake Assessment (NLA; 148 reservoirs) and the National Rivers and Streams Assessment (NRSA; 2121 rivers and streams) of the United States Environmental Protection Agency (USEPA), we evaluated the impacts of river impoundment on macroinvertebrate biodiversity at three spatial scales (i.e., reservoir, ecoregion and country scale). We used a space-for-time substitution approach to model the impact of impoundment (i.e., we used rivers and streams as the before-impoundment conditions, and reservoirs as the after-impoundment conditions). We expressed the impact on biodiversity in terms of potentially disappeared fraction of species (PDF) to be used in the life cycle assessment (LCA) framework. To understand the role of regionalization, and some potentially influential variables, on changes in macroinvertebrate richness following impoundment in the United States, we used analyses of variance (ANOVAs) as well as variation partitioning, and developed empirical predictive models. Overall, 26% of macroinvertebrate taxa disappeared following impoundment in the United States, and PDFs followed a longitudinal gradient across ecoregions (i.e., higher PDFs in the western part of the country, lower PDFs in the eastern part). We also observed that large and oligotrophic reservoirs, located in high elevation had high PDFs. This study provides the first empirical PDF values for macroinvertebrates to be used as characterization factors (CFs) by LCA practitioners. We also provide strong support for regionalization and a simple predictive model to be used by LCA modellers.