Abstract: Water stress and unfavorable climate decrease the growth and development of plant globally. Medicago is a valuable plant as a source of food for animal, foliage and for medicine. Since, Iran is located in a dry area, consequently, the study of plant responses to water stress is important. In this study UV-C radiation was used to induce physiological and genetic changes in alfalfa callus. In vitro grown calluses of M. sativa were exposed to UV-C for 0,15, 30 and 60 min and then, were transferred to MS medium containing 0, 2, 5, 10, 20 and 30% PEG. All explants were kept in the dark in the culture room. Calluses tolerate to osmotic stress were selected according to fresh weight. Results showed that radiation of UV-C for 60 min increased the osmotic tolerance and decreased the effect of drought stress.