Objective: This research aimed to evaluate the effects of hypoxia at different altitudes and durations on the expression of hypoxia-inducible factor-1α (HIF-1α) in rat tissues. Methods: A total of 72 Wistar rats were used to investigate the effect of hypoxia at different durations on rat tissues and 72 Wistar rats were used to investigate the effect of hypoxia at different altitudes. Hematoxylin and Eosin (HE) staining was performed to observe the pathological changes of hippocampus tissues, and the expression of HIF-1α of rats under conditions of hypoxia was detected by quantitative real-time polymerase chain reaction and western blotting. Results: According to the pathological results, we found that the degree of the brain, lung, and heart damage and HIF-1α expression, showed an increasing trend as the altitude (1,500, 3,000, 4,500, 6,000, 7,500, and 8,000 m for 12 hours) and duration (0, 6, 12, 24, 36, and 72 hours at 7,500 m) of hypoxia increased. Although there is a significant difference at 8,000 m, considering model stability, animal ethics and cost, we chose 7,500 m as a fixed altitude during hypoxia at different durations. Compared with the normoxic group, the expression of HIF-1α mRNA in the 7,500 m significantly increased by 30.48%, 21.00%, and 12.62%, in brain, lung, and heart tissue (p < 0.01), and HIF-1α mRNA in the 72-hour hypoxic exposure group significantly increased by 52.58%, 20.39%, 27.88% in tissues (p < 0.05). Compared with the normoxic group, HIF-1α protein expressions in the 7,500 m significantly increased by 10.26%, 31.71%, and 13.33% in brain, lung, and heart tissue (p < 0.01, p < 0.01, p < 0.05), and HIF-1α protein expressions in the 72-hour hypoxic exposure group significantly increased by 18.89%, 22.89%, and 29.75% in tissues (p < 0.05). Conclusion: HIF-1α expression in the rat was correlated with altitude and duration of hypoxic exposure.