Vitis amurensis (Shanputao) is the most cold tolerant Vitis species and so is of great interest to grape breeders and producers in areas with low winter temperatures. Here, we report its high-quality, chromosome-level genome assembly based on a combination of sequence data from Illumina and PacBio platforms, BioNano optical mapping and high-throughput chromosome conformation Capture (Hi-C) mapping. The 604.56-Mb genome contains 32 885 protein-coding genes. Shanputao was found to share a common ancestor with PN40024 (V. vinifera) approximately 2.17-2.91 million years ago, and gene expansion observed in Shanputao might contribute to the enhancement of cold tolerance. Transcriptome analysis revealed 17 genes involved in cold signal transduction, suggesting that there was a different response mechanism to chilling temperature and freezing conditions. Furthermore, a genome-wide association study uncovered a phosphoglycerate kinase gene that may contribute to the freezing resistance of buds in the winter. The Shanputao genome sequence not only represents a valuable resource for grape breeders, but also is important for clarifying the molecular mechanisms involved in cold tolerance.