A synthetic autopolyploid was developed from diploid Aegilops tauschii, D genome progenitor of common wheat (Triticum aestivum). The tetraploid Ae. tauschii displayed a markedly larger organ size than the diploid donor. Fluorescence in situ hybridization (FISH) and DNA marker analysis revealed that there is no clear variation at either the chromosomal or DNA level between the diploid and tetraploid plants. We analyzed the variation in cytosine methylation patterns between the diploid and tetraploid plants by methylation-sensitive amplified polymorphism (MSAP) and detected 228 and 232 methylated sites in diploid and tetraploid plants, respectively. Statistical comparison indicated that the tetraploid Ae. tauschii genotype displayed no significant difference in polymorphic methylation level compared to the diploid ones. Twenty-two different genomic fragments displaying different methylation behavior during the ploidy conversions were isolated and sequenced. It demonstrated that alterations in the level of methylation have the most profound effects on coding genes. We demonstrated that there are some genes expressions modified by DNA methylation may be correlated with phenotypic alteration after autotetraploidization.