Low temperature is one of the most widespread stress factors limiting productivity of cereals. Tolerance to low temperature was studied in 12 barley (Hordeum vulgare L.) cultivars with different growth habits (winter, spring, and facultative) subjected to low, non-freezing temperature (5°C) for cold acclimation, then exposed to freezing (‒5°C) treatment. Different phenotypic assays, including biomass production and survival rate (%) were conducted to determine the range of genotypic variation for tolerance to low temperature. Although the most tolerant cultivar was a winter/facultative one (‘Abidar’) and the most susceptible one was a spring cultivar (‘Nik’), little relationship existed between frost tolerance and growth habit when all 12 tested cultivars were compared. According to the data obtained, four cultivars with contrasting survival rate were selected and metabolic alterations were analyzed in these cultivars after cold acclimation and de-acclimation (25°C) treatments. Soluble carbohydrates and proline were accumulated in the leaves up to 20-70% (P< 0.05) and 37 fold (P< 0.001) during the acclimation period, respectively, and decreased upon de-acclimation by 9-42% (P< 0.05). In contrast to the total protein, proteins in the leaf apoplast, along with carbohydrates, accumulated up to 2-4 fold (P< 0.05) during the acclimation period. The frost tolerance of cultivars was correlated (r= 0.91, P< 0.05) with the concentrations of apoplastic carbohydrates but not with the total and apoplastic proteins or total carbohydrates. Our data revealed that barley cultivars were different in the metabolic adjustments upon acclimation treatment; some of these metabolic responses were directly associated with frost tolerance of cultivars.