[Objectives] Understanding seasonal and habitat-related variations in stress hormone levels is essential for assessing wildlife physiological responses and adaptation to environmental changes. The Przewalski’s Gazelle (Procapra przewalskii), a rare and endangered species endemic to China, has long been affected by habitat fragmentation and human activities. It holds significant research value for exploring the physiological response mechanisms of endangered species to environmental stress. This study aims to compare seasonal fluctuations in fecal cortisol levels between wild and semi-captive populations and investigate sex differences in stress responses. [Methods] Fecal samples were collected from wild gazelles in the eastern region of Qinghai Lake and semi-captive individuals at the South Bank Rescue Center across three seasons: summer (June–July), autumn (September–October), and winter (January). Cortisol concentrations were quantified by radioimmunoassay (RIA). Prior to analysis, Shapiro-Wilk tests and Levene’s test were performed to assess data normality and homogeneity of variance. Multivariate analysis of variance (MANOVA) was performed to examine the effects of habitat, season, and sex on cortisol levels, followed by Mann-Whitney U tests for pairwise comparisons. [Results] Cortisol levels varied significantly across seasons (F2, 155 = 48.222, P < 0.001) and between habitats (F1, 155 = 5.244, P = 0.023), with wild individuals exhibiting higher cortisol levels than semi-captive ones, particularly in autumn and winter. Seasonal fluctuations followed a distinct pattern, with peak cortisol levels observed in summer and the lowest levels in winter. A significant interaction between sex and season was detected (F2, 155 = 4.962, P = 0.008). In summer, females exhibited higher cortisol levels than males (U = 25.0, P = 0.013), whereas this trend became opposite in winter (U = 102.0, P = 0.023). The results are illustrated in Figs. 2 and 3. [Conclusion] This study provides the first quantitative comparison of stress hormone levels between wild and semi-captive Przewalski’s Gazelles, revealing distinct seasonal and sex-related variations. Elevated cortisol levels in wild populations during autumn and winter likely reflect heightened environmental stressors, such as food scarcity and harsh climatic conditions. Seasonal peaks in cortisol correspond to key biological events, with higher summer levels in females potentially associated with reproductive demands and heightened vigilance, while increased winter cortisol levels in males may be linked to mating competition. These findings improve our understanding of the physiological adaptation of this endangered species to environmental stresses and offer critical insights for formulating conservation strategies.