Abstarct: Fenugreek is a medicinal and winter herbal that contains a large amount of the active ingredient diosgenin. Diosgenin is one of the most important quality factors in fenugreek, which is widely used in the pharmaceutical industry as a raw material for the production of steroid drugs. The aim of this study was to investigate the effect of epi-brasinosteroid on exxpression of Diosgenin-related genes and some of the physiological properties of Fenugreek under high temperature stress. The treatments of this study consisted of epi-ebrosinosteroid (0, 2, 5 and 10 ppm) as well as Different harvest times (6 and 24 h) and High temperature stress (42° C) in two populations of Boshroyeh and Hamadan. The results showed that the pattern of CAS gene exxpression was similar in both populations. Gene exxpression of SEP was similar to CAS in both populations, although the exxpression of SEP gene in Hamadan was much higher compared to Boshroyeh. The exxpression of SMT gene was also similar in both populations, except that the exxpression of this gene in Boshroyeh increased almost 17-fold when applied to high temperature stress and 2 ppm level of brassinosteroid. The exxpression pattern of SSR in high temperature treatment and application of different levels of brassinosteroid was different between the studied populations, so that the exxpression of this gene in Hamadan at 2 ppm level was increased 3-fold compared to normal temperature. SQS gene exxpression was decreased in both populations after using high temperature treatment and different levels of brassinosteroid compared to normal conditions. Prolonged heat treatment (24 hours) increased the exxpression of the genes studied in this research more effectively than short-term heat treatment (6 hours). In the other part of the study, high temperature stress reduced the content of chlorophyll a, chlorophyll b, total chlorophyll, carotenoid and increased malondialdehyde. Application of epi-ebrosinosteroid during stress not only prevented the reduction of the above-mentioned traits but also increased them. Applying different levels of epi-ebrosinosteroid (2 and 5 ppm) at high temperatures resulted in decreased malondialdehyde and increased resistance to fenugreek, possibly due to the reduced free radical production and non-fatty acid peroxidation. The activity of Catalase, Guaiacol peroxidase and Ascorbate peroxidase enzymes increased in stress condition and application of 2 and 5 ppm epi-ebrosinosteroid, resulted in further increase of these enzymes activity under stress conditions. Overall, the results of this study showed that epi-ebrosinosteroid (2 and 5 ppm) might be used as a suitable stimulus to increase the resistance of fenugreek to heat stress. High temperature treatments, especially for 24 hours, and different levels of epi-ebrosinosteroid (2 and 5 ppm) can also be applied to increase the exxpression of some genes involved in the biosynthesis pathway and subsequently to increase the content of the biosynthesis.