Recent studies revealed that DNA methylation plays an important role in plant growth and development. In this study, a water-saving and drought-resistantricevarietyHuhan3wassubjected to drought stressfrom tillering to grain-filling stages insix successivegrowth cycles. The variations in DNA methylation pattern between the original generation (G0) and the sixth generation (G6) were analyzed by usingmethylationsensitiveamplificationpolymorphism method. The results revealed that themethylated loci accounted for34.3% to 34.8%ofthetotal loci. Amongthese methylated loci,83.1% to84.8% were full-and hyper-methylated and15.2% to16.9%were hemi-methylated. The DNA methylation level decreased from the three-leafto four-leaf stagesin Huhan3. Differentially methylated loci (DML) between generationsor/and between different developmental stages accounted for 4.0% of the total loci, most of which were only related to plant development (57.9%). Compared to G0, the DNA methylation pattern of G6changed after drought domestication, at the three-leafstage, de-methylation accountingfor59.1%, while atthefour-leaf stage, re-methylation for47.9%. Genome-wide alternations of DNA methylation were observed between the two seedling stages, and DML mainly occurred on the gene’s promoter and exon region. The genesrelated toDML involved in a wide range of functional biologyand participated in many important biological processes.