The purpose of this study was to investigate the periodic seasonal changes in endocrine activity and gonadal development of female rainbow trout (Oncorhynchus mykiss) in a high-altitude cold-water environment. The fish were sampled monthly from January to November and the levels of plasma hormones (estradiol (E₂), cortisol and thyroid hormones (TH_S)) and vitellogenin (VTG) were measured by ELISA. Moreover, the transcriptions of sex-related genes in the ovary, brain, and liver were detected by qRT-PCR. The results showed a seasonal fluctuation of plasma hormones and VTG together with the development of the ovary, which reached a peak from August to October. Similarly, the transcription of hypothalamic gonadotropin-releasing hormone-2 (cgnrh-2), hypothalamic gonadotropin-releasing hormone receptors (gnrhr) and follicle-stimulating hormone (fsh) in the brain varied from January to September, but the highest level was detected in September to November. In addition, the transcription of sex-related genes located in the ovary and liver increased significantly during August to October, accompanied by a continuous increase in the gonadosomatic index (GSI) and a decrease in the hepatosomatic index (HSI). Therefore, plasma hormones and sex-related genes regulate the development and maturation of O. mykiss oocytes with the change in seasons and peaked in November. The results of this study provide a reference for improving the efficiency of the artificial reproduction of O. mykiss.