Migrating adult Pacific salmon (Oncorhynchus spp.) are sensitive to warm water temperatures (> 18 °C) with population-level consequences including mortality prior to spawning along migration corridors (i.e., en route mortality) or spawning grounds (i.e., prespawn mortality). Warm temperatures are surprisingly common in the Yukon River across the 23-year record (1996–2019) with temperatures exceeding 18 °C in 20 years, 19 °C in 17 years, and 20 °C in 10 years. Given that warm water temperatures are routinely encountered by Yukon River Chinook salmon (O. tshawytscha) and the stock has not recovered since declines in the late 1990s, we examined the proportion of individuals with evidence of heat stress to assess the potential of freshwater adult mortality to contribute to population trends. Heat stress was identified using gene transcription and heat shock protein 70 biomarkers from muscle tissue in 2016¬ and 2017 informed by experimental validation. The combination of both biomarkers identified heat stress in more than half of Chinook salmon examined (54%, n = 477) across all capture locations and both study years. Chinook salmon collected in 2017 generally had higher proportions of heat stress compared to 2016 in agreement with warmer temperatures in the mainstem Yukon River (mean±SD July water temperature 18.7±0.98 °C vs 18.3±0.93 °C at Pilot Station). Our results suggest that warm water temperatures may be contributing to the failure of Yukon River Chinook salmon to recover. Widespread unusual mortality of migrating Pacific salmon during the record-breaking warmth and drought of 2019 provides additional evidence that river conditions will become increasingly important to Alaska’s Pacific salmon. This work provides actionable science to decision makers in the northern part of the Pacific salmon range where heat stress has not been considered a conservation and management threat and low in river mortality is assumed in escapement-based management goals.