There are growing concerns regarding the safe passage of aquatic organisms over large hydroelectric dams on major rivers like the Susquehanna River, as well as lesser barriers such as culverts. It is unknown if efforts to mediate this issue (e.g., increased stocking and fish passage facilities) are significantly effective at restoring populations of Anguilla rostrata. A. rostrata (American eel) faces population decline due to impediment of anadromous migration from the North Atlantic. They are major hosts of important native freshwater mussels such as Elliptio complanata (Eastern elliptio mussel) and are key food web members. Eels are cryptic organisms, which poses a challenge for traditional electrofishing sampling methods; environmental DNA (eDNA) is a popular method for detection of cryptic species. In the fall of 2025, we began an eDNA sampling initiative in the Susquehanna basin, targeting sites upstream and downstream of all major barriers on the main stem of the river and its tributaries. Samples were taken using the Smith-Root eDNA Sampler Backpack. We filtered ~1L (±0.10) of water through 0.45 µm filters. Filters were stored in ethanol until they were processed via DNA extraction and qPCR. DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit, and qPCR procedures were completed using a recently developed PCR marker for American eels referred to as AME1. The Bio-Rad CFX Opus 96 Real-Time PCR System was used for 15 μL qPCR reactions, and the CFX Maestro computer program was used to analyze PCR results. Water quality measurements were taken at each site to better our understanding on potential predictors of eel movement. Current findings indicate presence of eels downstream of major hydroelectric dams on tributaries feeding into the West Branch of the main river, but no presence around low head dams on the main river. More sampling will be completed in eastern and southern Pennsylvania, around the mouth in northern Maryland, as well as the headwaters in upstate New York. Understanding where eels are present as well as how they are moving will guide future stocking and conservation efforts, especially in relation to fish passage systems.