Video S1. Two Ocean Pass South of Yellowstone National Park, Wyoming. Two Ocean Pass connects the headwaters of the Atlantic and Pacific drainages in the Bridger-Teton Wilderness of Wyoming south of Yellowstone National Park. Here, a broad alpine meadow straddles the Continental Divide at 2,478 m elevation, and headwaters of the Columbia and Missouri drainages originate from a single perennial stream; North Two Ocean Creek flows along the Continental Divide and branches into Pacific Creek, a Snake River tributary flowing to the west, and Atlantic Creek, a Yellowstone River tributary flowing to the east. The pass is a nearly level meadow near the center of which is a marsh that becomes a small lake in times of wet weather or snowmelt runoff. No barrier prevents the movement and mixing of fish between Pacific Creek and Atlantic Creek. Following glacial recession from the region about 14,000 years ago, ancestral Yellowstone cutthroat trout colonized the Yellowstone River drainage from sources in the lower Snake River drainage over Two Ocean Pass. They dispersed downstream and were the only trout inhabiting Yellowstone Lake for thousands of years prior to the establishment of Yellowstone National Park. The watershed of the Yellowstone River upstream of Yellowstone Lake, including Two Ocean Pass, is among the most remote in the contiguous United States and lies largely within protected federal wilderness. In July 2019, Yellowstone National Park and Wyoming Game and Fish Department biologists, with assistance from Wyoming Trout Unlimited and the Wyoming Storer Foundation, sampled environmental DNA in the connected waters near Two Ocean Pass. The sampling was conducted to determine if invasive lake trout or other nonnative fish were present, and could thereby colonize the Yellowstone River basin in the past or future and threaten native cutthroat trout of Yellowstone Lake.