DNA methylation is one of the most important epigenetic processes that participates in the organization of chromatin structure and plays an important role in regulating gene expression. Sequence-related amplified polymorphism (SRAP) is a simple but an efficient gene amplification marker system for both mapping and gene tagging in plants. Combined methylation sensitive restriction enzyme digested genomic DNA with SRAP and methylation sensitive-sequence related amplified polymorphism (MSSRAP) marker system was developed to investigate de novo DNA methylation in the plant genome. To investigate the efficiency of this new marker system, DNA samples of 5 different tissues from 3 individuals of Brassica napus W10 (DH line) were employed for de novo DNA methylation analysis. Results indicated that approximately 0.2% reproducible polymorphic fragments were found among the five different tissues. Sequencing results demonstrated that some of these polymorphic fragments were matched well with the chloroplast encoding genes, photosynthetic related genes and the genes encoding protein with unknown functions in Genbank. Therefore, MS-SRAP marker system is a simple but efficient system for detecting gene de novo methylation.Key words: Brassica napu,de novo methylation,sequence related amplified polymorphism, methylation sensitive.