Nitrogen (N) fertilizer runoff into rivers is linked to nutrient enrichment, hydrologic alteration, habitat degradation and loss, and declines in biotic integrity in streams. Nitrogen runoff from agriculture is expected to increase with population growth, so tracking these sources is vital to enhancing biomonitoring and management actions. Unionid mussels are large, long-lived, sedentary, primary consumers that transfer particulate material and nutrients from the water column to the sediments through their filter feeding. Because of these traits, mussels may provide a temporal integration of nitrogen inputs into watersheds. Our goals were to (1) establish a baseline delta15N signature for unionid mussels in watersheds not heavily influenced by agriculture for use in comparative analyses and (2) determine if mussels provide an integrative measure of N sources in watersheds with varying percentages of agriculture across large spatial scales. We compiled tissue delta15N data for 20 species of mussels from seven geographic areas, including 23 watersheds and 42 sample sites that spanned varying degrees of agricultural intensification across the eastern United States and Canada. We used GIS to determine land cover within the study basins, and we estimated net anthropogenic nitrogen inputs (NANI) entering these systems. We then determined the relationship between mussel tissue delta15N and percentage of land in agriculture (%AG) and net anthropogenic N loading. The delta15N of mussel tissue could be predicted from both %AG and net anthropogenic N loading, and one component of NANI, the amount of N fertilizer applied, was strongly related to the delta15N of mussel tissue. Based on our results, mussels occupying a system not affected by agricultural land use would have a baseline delta15N signature of approximately 2.0 pe thousand, whereas mussels in basins with heavy agriculture had delta15N signatures of 13.6 per thousand. Our results demonstrate that mussels integrate anthropogenic N input into rivers at a watershed scale and could be a good bioassessment tool for tracking agriculture N sources.