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Simple Summary: It is widely accepted that climate change is a rapidly increasing threat to the aquaculture industry, with the biggest issue being a slow but continuous rise in water temperature and an associated decrease in dissolved oxygen in some areas. In New Zealand fish farms, during the hottest period of the year (January/February), increased water temperatures have been shown to be detrimental to Chinook salmon performance and are associated with mortalities. We report on the responses of Chinook salmon to a chronic, long-term increase in temperature and decrease in oxygen over a three-month period. We also identified, characterised, and developed relevant biomarkers for Chinook salmon and applied them to monitor responses to changes in the fish's environment. This paper provides evidence on how the use of molecular tools, alongside general health and haematological parameters, have the potential to better inform the aquaculture industry of stock health status, thereby enhancing resilience, efficiency, and productivity. In New Zealand, during the hottest periods of the year, some salmon farms in the Marlborough Sounds reach water temperatures above the optimal range for Chinook salmon. High levels of mortality are recorded during these periods, emphasising the importance of understanding thermal stress in this species. In this study, the responses of Chinook salmon (Oncorhynchus tshawytscha) to chronic, long-term changes in temperature and dissolved oxygen were investigated. This is a unique investigation due to the duration of the stress events the fish were exposed to. Health and haematological parameters were analysed alongside gene expression results to determine the effects of thermal stress on Chinook salmon. Six copies of heat shock protein 90 (HSP90) were discovered and characterised: HSP90AA1.1a, HSP90AA1.2a, HSP90AA1.1b, HSP90AA1.2b, HSP90AB1a and HSP90AB1b, as well as two copies of SOD1, named SOD1a and SOD1b. The amino acid sequences contained features similar to those found in other vertebrate HSP90 and SOD1 sequences, and the phylogenetic tree and synteny analysis provided conclusive evidence of their relationship to other vertebrate HSP90 and SOD1 genes. Primers were designed for qPCR to enable the expression of all copies of HSP90 and SOD1 to be analysed. The expression studies showed that HSP90 and SOD1 were downregulated in the liver and spleen in response to longer term exposure to high temperatures and lower dissolved oxygen. HSP90 was also downregulated in the gill; however, the results for SOD1 expression in the gill were not conclusive. This study provides important insights into the physiological and genetic responses of Chinook salmon to temperature and oxygen stress, which are critical for developing sustainable fish aquaculture in an era of changing global climates. [ABSTRACT FROM AUTHOR]