We have found that cauliflower mosaic virus (CaMV) 35S promoter-specific transgene silencing is mediated by DNA methylation in gentian (Gentiana triflora × G. scabra). De novo methylation of asymmetric cytosines (CpHpH; where H is A, C, or T) sequence has been detected at the enhancer region (−148 to −85) of the 35S promoter in transgenic gentians, and is thought to be responsible for the silencing mechanism. To clarify the concept of de novo methylation, the present study examined the detailed DNA methylation profile of the entire T-DNA sequence (ca. 4 kb) integrated into transgenic gentians. Although highly methylated cytosines at CpG and CpWpG (W is A or T) sequences were broadly distributed, except in the sGFP coding region, highly methylated cytosines at CpHpH and CpCpG sequences were mainly limited to the 35S enhancer region. In addition to the previously identified de novo methylation peak (−148 to −85), another peak was discovered at −298 to −241. Electrophoretic mobility shift assays showed that gentian nuclear extracts could bind to the corresponding probes (−149 to −124 and −275 to −250), and that the probes could compete with one another for binding. Thus, a nuclear factor might be involved in the de novo methylation of the two regions. In addition, the present data indicated that the methylation patterns at CpCpG sites could be categorized as CpHpH methylation rather than CpWpG methylation.