Waterborne transmission of Toxoplasma gondii is assumed to be enhanced in areas with human-altered landscapes (e.g., urbanization, agriculture) and increased populations of non-native domestic and feral cats (Felis catus). However, little is known concerning T. gondii exposure risks in more natural watersheds (e.g., reduced human footprint, no domestic or feral cats) to establish a baseline for comparisons. In this study, muskrats (Ondatra zibethicus) were used as sentinels to assess baseline T. gondii exposure in a relatively pristine watershed in the Greater Voyageurs Ecosystem, northern Minnesota, during the summers of 2018–2019. Toxoplasma gondii antibodies were assayed in sera of live-trapped muskrats (n = 70) using a modified agglutination test. None of our samples were positive for T. gondii antibodies (P = 0.00, 95% Wald Score Confidence Interval = 0.00–0.05). This study establishes a baseline to compare T. gondii waterborne transmission risks in other human-modified watersheds.