Risk of weather variability is amongst factors that always affect the cereal production in many areas. In order to determine the freezing tolerance of five barley (Hordeum vulgar L.) cultivars under controlled conditions, a factorial experiment based on CRD carried out with three replications at the crop physiology Laboratory of College of Agriculture, Ferdowsi University of Mashhad. Treatments were barley genotype with five levels, including Makouei, Karun×Kavir, lokht, Reyhan and Valfajr, and temperature in six levels (0, -4, -8, -12, -16 and -20 °C). Plants were kept under natural conditions until 4-6 leaf stage to provide the acclimation period and then were transferred to a thermo-gradient freezer to apply freezing stress. The cell membrane stability of genotypes was measured through electrolyte leakage (EL) and the LT_50el (temperature for killing 50% of samples according to the electrolyte leakage) was also determined. Furthermore, survival percentage, plant height, dry weight, LT_50su (temperature for killing 50% of samples according to survival) and RDMT₅₀ (reduced dry matter temperature) were determined after three weeks recovery in the glasshouse. Results showed that leakage percentage from leaf and crown after freezing was significantly different (P<0.01) amongst cultivars. The greatest (69.4) and the lowest (58.5) leakage percentage from leaf samples were observed in Valfajr and Karun×Kavir cultivars, respectively. Reyhan cultivar showed the highest (78.9) and Makouei, Karun×Kavir, lokht and Valfajr cultivars showed the lowest leakage percentage from crown samples. Plant height, dry weight and LT_50su of cultivars were significantly different after freezing. For instance, the dry weight of plants after three weeks recovery was 978 and 480 mg/plant for Karun×Kavir and Valfajr cultivars, respectively. Most studied traits were significantly reduced under temperatures lower than -12^oC compared to the control.