DNA methylation plays an important role in the growth and development of plant, and would change under different environments. In this study, 5-methyl cytosine (5mC) content and methylation level exhibited tissue specificity in genomic DNA of wheat seedling, and increased significantly in leaf along with the increase of water deficit, which was especially significant in leaf of wheat AK58. Full-methylation might dominate in genomic DNA of wheat seedling, the increase of full-methylation level under water deficit was significantly higher than that of hemi-methylation level. Under water deficit, DNA methylation of wheat seedling showed significant polymorphism, this polymorphism was always higher in root, especially was higher in root of wheat AK58. Further analysis appeared that changes of DNA methylation in wheat seedling took methylation as principle and demethylation as supplement under water deficit. Therefore, under water deficit, the degree, level and polymorphism of DNA methylation in wheat seedling showed tissue specificity and species specificity, and were higher in wheat AK58 compared with those of wheat XM13, perhaps wheat AK58 could more rapidly respond to water deficit by changes of DNA methylation, which would contribute to reveal molecular mechanism of wheat adapting to water deficit.