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In order to study the freezing resistance of some barley varieties using rates of electrolyte leakage this experiment was carried out on three barley variety, including Reyhan, kavir, and Nosrat. These varieties are the most important varieties that cultivated in Semnan province. For this experiment, barely seeds were sown in small containers. The current experiment was carried out as Factorial experimental design plan based on completely randomized design (CRD).The first factor in this study was barley variety in three levels. The second factor was temperature in 5 levels including +4°C, -4°C, -8°C, -12°C and -15°C. Seedlings in the stage of tillering, was sprayed by water and then were put in the incubator for two hours. Then two gram samples was selected and 20 mm deionized water was added and shakes for 24 hours. Then every day for a week EC in solution was measured. Analysis of variance (ANOVA) was used to determine significant (p < 0.01 and p < 0.05) differences between barley genotypes. The differences between means of barley varieties were inspected using Duncan test . The results showed that the percentage of ion leakage between the three cultivars had significant differences .The effect of temperature on the percentage of ion leakage in all days, had significant difference.The highest percentage of ion leakage measurements on all days was at the -15°C and the lowest percentage of leakage occurred at 4 °c . Kavir variety in comparison with other varieties had lower cold tolerance. Interaction of temperature and genotype factors on ion leakage measurements showed that all varieties had significant differences.