Abstract: DNA methylation is one of the most important mechanisms in the epigenetic regulation in eukaryotic cells. DNA methylation affects gene expression and plays an important role in plants adapting to adverse environments. Tall fescue (Festuca arundinacea), a gramineous monocot, has superior resistance to cold and heat. It is widely used for sports turf, erosion control, and as a forage grass. However, the distribution and yield of tall fescue is constrained by drought stress. In this paper, the growth was measured and the DNA methylation profile of tall fescue (cv. Barlexas) evaluated using methylation-sensitive amplification polymorphism (MSAP) after 15 d drought treatment. The growth of the plants was seriously restricted under water-deficit challenge. Genome-wide, a total of 475 CCGG sites were detected by MSAP using 10 selective primer pairs in tall fescue seedlings. Among these sites, 131 sites (27.58%) showed drought-induced methylation and demethylation. The total methylation level was 43.16% in control and 42.11% in drought-treatment, which indicates a 1.05% decrease when exposed to drought. The sequences of 13 amplified fragments were successfully obtained, representing the differential types of methylation changes. Homologous analysis showed that most of the fragments were relevant to stress. Two sequences (Fa6 and Fa7) with enhanced methylation induced by drought were homologous to a retrotransposon of Triticum aestivum and Hordeum vulgare, respectively, showing drought induced methylation of the retrotransposons. The methylation of transposons is vital in maintaining DNA stability. In summary, drought stress decreased the total DNA methylation level in the tall fescue genome, and the altered methylation in response to stress was probably involved in environmental stress acclimation.