Temperature is crucial for fish physiology, as metabolism and related physiological processes are directly influenced by thermal energy. Against the backdrop of global warming, climate-induced variations in water temperature increasingly constrain fish physiology. Consequently, understanding the effects of temperature on fish metabolism is vital for predicting how global warming might impact various fish species. Plateau fish, predominantly cold-water species, exhibit greater sensitivity to temperature fluctuations. However, research on the effects of temperature on plateau fish is currently limited. Consequently, this study employed low-altitude H. nobilis as a reference while D. macrophthalmus and A. grahami were selected from the Yun–Gui Plateau. Following 15 days of temperature acclimation at 10, 15, 20, 25, and 30 °C, organ mass, the resting metabolic rate, and mitochondrial function were measured. The results indicated that high-altitude fish exhibit heightened metabolic sensitivity, demonstrating more pronounced increases or decreases in metabolic rates as temperature increases, along with limited plasticity in organ size. This may render high-altitude fish more vulnerable to the impacts of climate warming. Furthermore, physiological differences between altitudes and species were observed, primarily characterized by higher metabolic rates across all measured temperatures in plateau species. Additionally, plateau fish presented greater masses of heart, red muscle, and liver but smaller masses of brain and kidney. We propose that the trade-off between elevated metabolic rates and organ size may represent an adaptive strategy for fish inhabiting high-altitude environments, involving specific ecological costs and benefits. These findings not only address the knowledge gap regarding the metabolic characteristics of fish on the Yun–Gui Plateau but also provide theoretical and experimental foundations for the conservation of high-altitude fish populations.