Low temperature stress, cause many physiological and biochemical process in plants. Different genotypes of grapes show diverse reaction to cold stress and therefore so, the selection of cold tolerant genotypes is the most effective method to avoid frost damages. In vitro culture is efficient methods for selecting cold tolerant plants. Callus was formed in three grapevine genotypes; including Bidaneh Sefid, Dom-rubahi and Askari, and then callus were incubated for cold accilimation, a period of 24 days in darkness at 4°C. The contents of soluble sugar, proline, dry weight precentage and the ploid level in acclimated and control callus have been measured. Also, survival test at different freezing temperatures were conducted. Maximum callus induction was obtained in the medium containing 5 and 10 µM of 2, 4-D and 5 µM NAA in combination with 1 µM BA. In the Askari genotype, in terms of low temperature, low proline content was observed. By making cold acclimation, proline, dry matter and total protein increased that, the increase in Bidaneh Sefid and Dom-rubahi was more than Askari. The results of this study suggested that the three genotype understudy, Bidaneh Sefid, showed greater resistance to freezing temperatures, as well as the effect of cold on ploidy level genotypes, stable ploidy level and genetic stability was observed, and that is the most important based on the in vitro micropropagation and proliferation of studied genotypes.