Fragmentation of riverine habitat by anthropogenic barriers is one of the primary threats to aquatic species in the United States. However, the degree of fragmentation of aquatic habitat from undersized and perched road crossing structures is largely unknown. Over two million road crossing structures exist in the Southeastern United States, yet only 40,000 have been assessed to determine if they are barriers to aquatic organism passage. Since 2015, as a part of its Aquatic Connectivity Program, the Southeast Aquatic Resources Partnership (SARP) has been leading an effort to collect data using a standardized protocol developed by the North Atlantic Aquatic Connectivity Collaborative (NAACC). Since that time, over 300 people from a variety of partner organizations have been trained to use this protocol and have collected data at over 10,000 road crossings using SARP’s field application and Aquatic Barrier Inventory. These assessments have resulted in the replacement of more than 20 road crossing structures as well as a better understanding of the impacts of road stream crossing structures on aquatic organisms and their habitats across the region.