It is unclear if environmental DNA (eDNA) methods can be used for routine monitoring of the invasive Sea Lamprey (Petromyzon marinus). Previous research demonstrated that eDNA can detect invasive Sea Lamprey in both lab and field settings. However, the eDNA methods had not been optimized and validated for routine application. Three experiments were conducted to compare methods of eDNA field sample collection and create an optimized protocol. Then, eDNA sampling was benchmarked against standardized electrofishing at 54 sampling stations on 12 Great Lakes tributaries. eDNA detections (presence/absence) matched electrofishing detections at 79.6% of sampling stations. eDNA methods detected the novel presence of Sea Lamprey at 18.5% of sampling stations, and eDNA failed to detect electrofishing verified Sea Lamprey presence at 1.9% of sampling stations, demonstrating that Sea Lamprey detections from eDNA methods are highly congruent with detections from standard electrofishing protocols.