Natural radiogenic isotopes (primarily ⁸⁷Sr/⁸⁶Sr) from hot springs in the Upper Geyser Basin of the Yellowstone Plateau volcanic field and associated rocks were used to evaluate groundwater flow patterns, water-rock reactions, and the extent of mixing between various groundwater sources. Thermal waters have very low uranium concentrations and ²³⁴U/²³⁸U activity ratios near 1.0, which limit their utility as tracers in this reducing setting. Thermal waters have higher Sr concentrations (<22 ng/g) and a wide range of ⁸⁷Sr/⁸⁶Sr values that vary both temporally at individual discharge sites and between adjacent springs, indicating that conduits tap different subsurface reservoirs to varying degrees. Sr from local rhyolites have ⁸⁷Sr/⁸⁶Sr compositions that bound the range of values observed in groundwater throughout the basin. Non-boiling springs on the west flank of the basin discharge water with low ⁸⁷Sr/⁸⁶Sr consistent with flow through young volcanic rocks exposed at the surface. Boiling springs in the central basin have higher ⁸⁷Sr/⁸⁶Sr values reflecting interactions with older, more radiogenic volcanic rocks. Variability in upwelling thermal waters requires mixing with a low ⁸⁷Sr/⁸⁶Sr component derived from young lava or glacial sediments, or more likely, from deeper sources of hot groundwater circulating through buried Lava Creek Tuff having intermediate ⁸⁷Sr/⁸⁶Sr. Isotope data constrain basin-wide output of thermal water to 110–140 kg·s⁻¹. Results underscore the utility of radiogenic Sr isotopes as valuable tracers of hydrothermal flow patterns and improve the understanding of temperature-dependent water-rock reactions in one of the largest continental hydrothermal systems on Earth.