DNA methylation, an important epigenetic modification, serves as a key function in the polyploidization of numerous crops. In this study, early generations of resynthesized Brassica napus (F₁, S₁–S₃), ancestral parents B. rapa and B. oleracea were analyzed to characterize their DNA methylation status during polyploidization, applying DNA methylation-sensitive amplification polymorphism (MSAP) and high-performance liquid chromatography methods. In F₁, 53.4% fragments were inherited from both A- and C-genomes. Besides, 5.04 and 8.87% fragments in F₁ were inherited from A- and C- genome, respectively. 5.85 and 0.8% fragments were newly appeared and disappeared in resynthesized B. napus, respectively. 13.1% of these gene sites were identified with methylation changes in F₁, namely, hypermethylation (7.86%) and hypomethylation (5.24%). The lowest methylation status was detected in F₁ (38.7%) compared with in S₁–S₃. In S₃, 40.32% genes were methylated according to MSAP analysis. Sequencing of methylated fragments indicated that genes involved in multiple biological processes were modified, including transcription factors, protein modification, and transporters. Expression ananlysis of DNA methyltransferase 1 and DNA methyltransferase chromomethylase 3 in different materials was consistent to the DNA methylation status. These results can generally facilitate dissection of how DNA methylation contributes to genetic stability and improvement of B. napus during polyploidization.