Grapevine is one of the important small fruit crops in Iran and other parts of the world that in terms of food and pharmaceutical is of more importance. This plant has a wide ecological range of adaptability that can be seen from equator to the north and south regions of the earth. Grapevine by deep roots and mechanisms of tolerance, able to endure somewhat abiotic stresses (drought and salinity). In recent years, low rainfall and low water availability for agriculture has led to the identification and selection of genotypes and tolerant cultivars. One method of evaluation and selection of drought tolerant cultivars is the use of in vitro techniques that simulate controlling environmental stressors in vitro and pave the way, to more concisely study plant responses to drought stress. The present study was carried out in frame of factorial (5 x 4) completely randomized design with three replicates to investigate the responses of four Iranian grape genotypes “Askari”, “keshmeshi Ghermez Qazvin ”, “Yaqooti sefid” and “Shahani”) to deficit water stress, induced by polyethylene glycol (PEG) in in Vitro conditions. Micro-cuttings with length of 1 -1.5 cm of grapevine genotypes were prepared, disinfested and planted in the MS stock medium supplemented with 0.7 mg indole butyric acid (IBA). After rooting, microcuttings were transferred to MS medium containing various percent of polyethylene glycol (0, 3, 6, 9 and 12). Results showed significant reduction in all measured vegetative parameters including fresh and dry weight of shoot and root, shoot length, number of nodes per shoot and leaf area exposed to different levels of drought treatments. There were significant differences in the vegetative characteristics of the genotypes under study. The highest mean of the fresh weight of the shoot was related to genotype (Yaqooti sefid) and the lowest one to genotype (Askari), respectively. The shoot length of the Askari and Yaqooti Sefid genotypes was more in comparison with other two genotypes Ghermez Qazvin and “Shahani. Askari genotype had the highest leaf area, while Yaqooti had the lowest one. The highest mean of the root fresh weight was observed in Shahani genotype. In terms of root dry weight; there was no any significant difference among the genotypes. All the physiological parameters, like relative water content, leaf proline content, lipid peroxidation(MDA content) and ion leakage were affected by drought treatments. With increasing drought levels, leaf relative water content decreased and ion leakage, proline and MDA, were increased. Askari genotype had the largest and more relative water content than other genotypes. There was no significant difference among other genotypes in this respect. Ghermez Qazvin and Yaqooti Sefid genotypes had the highest rate of ion leakage, while that was lowest for Shahani. The highest and lowest amounts of MDA were related to Shahani and keshmeshi Ghermez Qazvin genotypes, respectively. Activities of antioxidant enzymes are also significantly affected by different levels of osmotic potential, so that with increasing levels of PEG, increases in activity of catalase, peroxidase, and ascorbate peroxidase were observed in the genotypes under study. Shahani genotype indicated more antioxidant activity than other genotypes and Askari was the later one. There was a significant negative correlation among morphological growth parameters with proline, ion leakage, and amount of malondialdehyde (MDA) and the activity of antioxidant enzymes. With respect to the results of this study, particularly of the physiological parameters and activities of antioxidant enzymes, could be concluded that, genotypes Shahani, Keshmeshi Ghermez Qazvin and Yaqooti Sefid, and Askari showed more, less and moderate tolerance to water deficit stress, respectively.