The presence of endocrine disrupting compounds (EDCs) exhibiting estrogenic activity in aquatic environments has been recognized as a widespread, pervasive environmental issue since the mid-1990s. These estrogenic compounds have been shown to adversely impact fish species at exposure concentrations as low as 1.0 ng/L Estradiol Equivalents (EEQ) (0.00367 nM). The aim of this research was to screen for all three fish estrogen receptors using the TriFishER assay to assess risk of impacts of estrogenic EDCs along the entire New River (360 miles) and link estrogenic EDCs to land use by screening for binding activity to three estrogen receptor (ER) isoforms (acERα, acERβa, acERβb) using the TriFishER assay. Results show that at most sites the estrogenic EDC concentrations represented a high risk for all 3 ER isoforms, despite collection of samples during 90th percentile river flows from seasonal rainfall. The acERα and acERβa were positively correlated to urban land use in stream sites, and acERβa was negatively correlated to forested land use. Since 23% of sites that showed undetectable binding to acERα simultaneously showed binding to acERβa and/or acERβb, the present study demonstrated the importance of screening for all three ER isoforms, as opposed to the customary screening for acERα. Tuberty is Professor and Assistant Chair of Biology at ASU, teaches Ecotoxicology and Zoology courses, and leads research on impacts of EDCs, coal ash, and other toxins to fish and invertebrates. He serves on the New River Conservancy’s Technical Advisory Committee and hosted the 2019 New River Symposium at ASU.