Abstarct: To assess the ecophysiological response of fenugreek ecotypes to late season water stress under biochar and non-biochar application, a greenhouse and a field experiment were conducted at the Agricultural Biotechnology Research Institute of Iran, Karaj, Iran in 2015. Six branched fenugreek ecotypes including Dezful, Shushtar, Ardestan, Rehman, Yazd, and Khomeyni Shahr were treated by biochar application and non-application under 2 different irrigation regimes of normal irrigation and water stress. A factorial experiment in a completely randomized design with 4 replications and a split plot factorial experiment in a randomized complete block design with 3 replications (Irrigation as the main plot and biochar and ecotype combination as the subplot) were used in greenhouse and field, respectively. Biochar was derived from rice husk at 500°C in an oxygen depleted environment and used mixed with the soil in pots in the greenhouse or top laxyer of the soil in the field at a ratio of 8:92 v/v. Water stress was applied on 50% flowering stage baxsed on %30-50 of soil field capacity in the greenhouse or increasing irrigation intervals from 4 to 8 days in the field. Yield, yield components and some compounds in seeds were measured in both experiments. According to the results, water stress significantly had a negative effect on growth and yield of fenugreek in both experiments. While using biochar moderated the effect of water deficit and decreased the effect of water stress on fenugreek growth and yield. It was shown biochar application increased seed yield under normal irrigation and water stress condition by 15.41 and 8.35%, respectively, in the greenhouse and by 24.33 and 26.66%, respectively, in the field. Among assessed ecotypes, Rehnan was superior to the other ecotypes from seed yield point of view in both experiments. Seed trigonelline content responded differently to biochar application in different irrigation regimes in both experiments. Overall, biochar application decreases secondary mextabolites accumulation in plants which are usually accumulate during stress periods by improving soil water content and decreasing water stress effect on plant. On the other hand biochar application increase trigonelline yield by increasing yield under water stress condition.