Introduction: Water scarcity is one of the major limiting factors to agricultural production across the globe and it is predicted that drought spells will occur more frequently in the future due to the long-term effects of global warming (Rivero et al., 2007). This has led to a decrease in canopy size, loss in photosynthesis and consequently reduced crop yields. Alfalfa, being perennial and multi cut, as well as having an efficient root system, can tolerate water limited conditions by dormancy and is able to restart its biological activities after irrigating (Mc Williams, 2002). The mechanisms of response to water stress are complex and include activation of growth regulators in molecular and physiological levels. In addition, the yield potential and stability of some Iranian alfalfa ecotypes in different locations have been investigated previously (Mofidian & Moghaddam, 2013). Material and Methods: The experiment was carried out with 10 warm and cold region alfalfa ecotypes in two distinct environments at Seed and Plant Improvement Institute (SPII), Karaj, Iran, over the years 2016-2018. The environments included full irrigation and no watering for 20 days from 22th of June. To conduct a preliminary evaluation of the ecotypes response to water stress, stress susceptibility index (SSI) based on fresh forage yield was calculated. The most important physiological traits measured in this study were as follows: relative water content (RWC), chlorophyll content index (CCI), canopy temperature depression (CTD), and stomatal resistance (SR), Chlorophyll a and b contents and carotenoids. Combined analysis of variance and mean comparisons were done by SPSS Ver. 22 and Excel 2016. Results and Discussion: The effect of no watering stress on all of the physiological traits was significant (P<0. 01). The ecotypes showed a significant difference in dry matter yield and all physiological measurements except for electrolyte leakage and chlorophyll b content. The year effect indicated a significant difference for chlorophyll b (P<0. 05); carotenoids and dry matter yield (P<0. 01) but it was not found to be significant for the rest of traits. Among the interactions, environment × year interaction showed no significant effect for all the studied traits. Although, ecotype × environment interaction was significant just for carotenoids (P<0. 01), ecotype × year interaction was significant for Dry matter yield, RWC, CTD and SR (P<0. 01); and CCI, chlorophyll a (P<0. 05), as well. Under full irrigation regime and cut-off irrigation treatment, RWC was respectively 66. 26 and 44. 06 percent, which indicated that water stress decrease RWC by 32 percent. Electrolyte leakage averaged 26. 51 percent under the stress environment, exhibiting a 72 percent decrease in cell membrane stability relative to full irrigation scenario. The chlorophyll content index in non-stress condition was 38 percent greater than that of under stress condition. The canopy temperature depression decreased by 43 percent during water stress imposition. No water shortage treatment led to the least stomatal resistance of 22. 22 s/cm; however, this value was 37. 87 s/cm under water-limited conditions, which showed a difference of 70 percent. Conclusion: The minimum stress susceptibility index at the first and second year, 0. 35 and 0. 51, were observed in BAGHDADI and BAMI ecotype respectively, which, among the investigated alfalfa ecotypes, were found to be the most resistance to no watering for 20 days. Overall, KFA6 ecotype showed the minimum electrolyte leakage (13. 79 s/cm) and stomatal resistance (23. 69 s/cm), respectively. Moreover, KFA6 ecotype had the maximum photosynthetic pigments of 1. 623 mg in fresh leaf (gr) that led to the production of 17. 48 t/ha dry matter yield and superiority of 14 percent as compared to the two-year mean of all ecotypes under full irrigation and no watering conditions. Keywords: Canopy temperature, stomatal resistance, cell membrane stability, photosynthetic pigments, and alfalfa. References: Mc Williams, D. 2002. Drought strategies for alfalfa. Extension handbook, Department of Extension Plant Sciences, New Mexico State University, Las Cruces, New Mexico. P-1-5. Rivero, R. M., Kojima, M., Gepstein, A., Sakakibara, H., Mittler, R., Gepstein, S., and Blumwald, E. 2007. Delayed leaf senescence induces extreme drought tolerance in a fl owering plant. Proceeding of National Academic Science, USA 104: 19631– 19636. Mofidian, S. M. A., and Moghaddam, A. 2013. Analysis of ecotype location interaction in cold-region alfalfa ecotypes. Iranian Journal of Crop Sciences, 15(2): 71-85.