Environmental DNA (eDNA) analysis is widely used as a low-impact, low-cost method for detecting sensitive species in aquatic systems. The application of eDNA techniques has been vital in a variety of biomonitoring, particularly with efforts to detect the presence of endangered or invasive species. While the fate of eDNA within lentic systems can be correlated with species abundance, there is greater difficulty interpreting eDNA “hits” in lotic environments. Moreover, successful mesocosm studies on eDNA decay and transport have brought to light the need to repeat these experiments in natural systems. Therefore, we describe the “DISTANCe” project, which aims to understand how abiotic factors, including pH, turbidity, water chemistry, and temperature, impact how far eDNA is transported in a natural stream. Using a method like constant-rate nutrient additions, we plan to conduct monthly eDNA injections of Atlantic Herring (Clupea harengus) material at the Mayfield Creek aquatic National Ecological Observatory (NEON) sites of central Alabama. We will establish rates of eDNA degradation and transport and leverage available NEON data to determine the dominant drivers of eDNA fate. We will replicate our experimental approach at NEON sites across multiple ecoregions of the contiguous U.S. Overall, the DISTANCe project will establish the connections between stream properties and the fate of eDNA, which remains a significant challenge for its effective use and interpretation.