Climate change is causing large scale declines in Pacific salmon, and warming rivers are causing high levels of en route mortality in spawning adults. In the Fraser River in British Columbia, Canada, only two of the thirteen Chinook salmon (Oncorhynchus tshawytscha) populations evaluated by COSEWIC were not assessed as threatened or endangered. However, little is known about the thermal tolerance of adult migrating and spawning Chinook salmon, and if differences exist among populations in physiological performance. We compared thermal performance of maturing Chinook captured from their spawning migrations from two populations: coastal fall-run Chinook (~100 km cooler migration) and interior summer-run Chinook (~500 km warmer migration), by measuring resting and maximum metabolic rates, aerobic scope (AS), and post-exercise recovery during acute exposure to current and future projected Fraser River temperatures (12, 18, 21, 24°C) encountered during their spawning migration. The temperature threshold for 50% mortality was 21°C for the coastal Chinook and 23.5°C for the interior Chinook, indicative of a lower thermal tolerance in the coastal population. Although both populations shared an optimal AS at 14.5°C, AS declined more rapidly with increasing temperature in the coastal population. Finally, while post-exercise recovery was impaired in both populations at high temperatures, the coastal population had a slower recovery compared to the interior population. Our findings suggest that migrating and spawning adult Chinook are vulnerable to environmental warming and display among-population variability in thermal performance.