Identifying early gene expression responses to hypoxia (i.e., low dissolved oxygen) as a tool to assess the degree of exposure to this stressor is crucial for salmonids, because they are increasingly exposed to hypoxic stress due to anthropogenic habitat change, e.g., global warming, excessive nutrient loading, and persistent algal blooms. Our goal was to discover and validate gill gene expression biomarkers specific to the hypoxia response in salmonids across multi-stressor conditions. Gill tissue was collected from 24 freshwater juvenile Chinook salmon (Oncorhynchus tshawytscha), held in normoxia [dissolved oxygen (DO) > 8 mg L-1] and hypoxia (DO = 4‒5 mg L-1) in 10 and 18°C temperatures for up to six days. RNA-sequencing (RNA-seq) was then used to discover 240 differentially expressed genes between hypoxic and normoxic conditions, but not affected by temperature. The most significantly differentially expressed genes had functional roles in the cell cycle and suppression of cell proliferation associated with hypoxic conditions. The most significant genes (n = 30) were selected for real-time qPCR assay development. These assays demonstrated a strong correlation (r = 0.88; p < 0.001) between the expression values from RNA-seq and the fold changes from qPCR. Further, qPCR of the 30 candidate hypoxia biomarkers was applied to an additional 322 Chinook salmon exposed to hypoxic and normoxic conditions to reveal the top biomarkers to define hypoxic stress. Multivariate analyses revealed that smolt stage, water salinity, and morbidity status were relevant factors to consider with the expression of these genes in relation to hypoxic stress. These hypoxia candidate genes will be put into application screening Chinook salmon to determine the identity of stressors impacting the fish.