Multiple rulemaking iterations have led to variable definitions of the “Waters of the United States” (WOTUS), a rule that determines which waterbodies receive federal protection under the Clean Water Act. The rulemaking process has incorporated American Indian Tribes as “stakeholders” rather than as sovereign peoples, compounding a colonial legacy that limits the ability of Indigenous peoples to choose appropriate strategies for water protection on Tribal lands. For example, protecting waters for Tribal beneficial uses requires applying both Western science and Indigenous knowledge to document patterns of waterbody connectivity and permanence, which underpin WOTUS policy. To honor the federal trust responsibility (a legal obligation) of the US Government to Tribes, policy should incorporate a parallel set of scientific standards for determining WOTUS on Tribal lands. These standards must recognize culturally distinct uses of waters and account for place-based Indigenous knowledge. Examination of the intersection of the science supporting water protection, Indigenous sovereignty, and US policy has relevance to similar issues around the globe.