Cold-water fishes, such as Brook trout (Salvelinus fontinalis), are being challenged by the consequences of climate change. The ability of these fish to acclimate to warmer environmental conditions is vital to their survival. Acclimation to warmer water may allow brook trout to reduce the metabolic costs of higher temperatures. Previous work has shown that brook trout display a significant thermal acclimation response in their myotomal muscle, with slower contractile properties observed in warm acclimated fish. In this study, gene expression was examined in hatchery brook trout acclimated to a range in temperatures (4, 10 or 20°C). Brook trout displayed variations in gene expression in their myotomal muscle in accordance with acclimation temperature. Genes important for muscle function, cellular metabolism, protein degradation, and stress response showed variation to both warm (20°C) and cold (4°C) acclimation. The warm acclimated fish also showed decreased expression of genes associated with aerobic metabolism and increased expression of genes for heat shock proteins, while the cold acclimated fish showed increased expression of genes associated with lipid metabolism and protein turnover. α-tubulin displayed a close association with thermal acclimation, increasing in expression with acclimation temperature. The patterns of muscle gene expression were the opposite of what was expected. Although warm acclimated fish have previously been shown to display slow muscle contractile properties, this study found that warm acclimation is associated with increased expression of genes for kinetically faster isoforms of important muscle proteins. Collectively, the results demonstrate a robust response to elevated temperature in the hatchery fish greater than 10,000 genes showing differential expression with temperature. These results provide a roadmap for the analysis of the acclimation response of native populations of brook trout encountering climate change.