Migrating adult Pacific salmon (Oncorhynchus spp.) are sensitive to warm water (>18 °C), with a range of consequences from decreased spawning success to early mortality. We examined the proportion of Yukon River Chinook salmon (O. tshawytscha) exhibiting evidence of heat stress to assess the potential that high temperatures contribute to freshwater adult mortality in a northern Pacific salmon population. Water temperatures greater than 18 °C have occurred almost annually in the Yukon River and correspond with low population abundance since the 1990s. Using gene transcription products and heat shock protein 70 biomarkers validated by field experiment, we identified heat stress in half of Chinook salmon examined (54%, n = 477) across three mainstem locations and three tributaries in 2016–2017. Biomarkers tracked wide variation in water temperature (14–23 °C) within a tributary. The proportion of salmon with heat stress differed between years at four of the six locations, with more prevalent heat stress in the warmer year. This work demonstrates that warming water temperatures are currently affecting northern populations of Pacific salmon.