Graphical Abstract The genomic landscape of cold-tolerant winter rapeseed ( Brassica napus , L) has been poorly characterized. We assembled a high-quality reference genome of a prominent cold-tolerant winter rapeseed cultivar, NTS57, and performed phylogenetic and pan-genomic analyses by integrating other reported Brassica napus accessions. The transcriptome analysis revealed that microtubule-associated biological pathways were much more active in NTS57 under cold stress than in the cold-sensitive variety. Whole genome methylation data analysis revealed that DNA demethylation on protein-coding genes and repetitive elements, especially at CHH sites, is essential for cold response in winter rapeseed.