The quality and yield of wheat (Triticum aestivum L.) are dramatically affected by drought. We used morphological and physiological characteristics and degree of DNA methylation to compare the responses of two wheat cultivars under osmotic stress, and found that the two cultivars behaved differently. Root development, leaf growth, and the accumulation of proline and soluble carbohydrate in wheat cv. AK58 all showed drought tolerance. Drought tolerance of wheat cv. XM13 was mainly improved by accumulation of proline and soluble carbohydrate. The degree of DNA methylation in wheat showed tissue specificity and increased significantly in leaf tissue with increasing osmotic stress, but decreased significantly in root tissue under mild osmotic stress. In addition, changes of DNA methylation differed between two wheat cultivars under osmotic stress, and this change was especially significant in AK58. Therefore, wheat AK58 may have stronger self-adjustment ability under osmotic stress compared with XM13, and might respond more rapidly to osmotic stress through the change of DNA methylation. This finding could be significant for revealing drought-tolerance mechanisms of plants. In this research, morpho-physiological characters and DNA methylation were used to study the responses of two wheat cultivars to osmotic stress. Two wheat cultivars differed in root development and leaf growth under osmotic stress, and AK58 showed significant changes in degree of methylation, which could help to reveal molecular mechanism of wheat adapting to osmotic stress from the perspective of epigenetics. [ABSTRACT FROM AUTHOR]