The burbot (Lota lota) is the only member of the cod family (Gadidae) that is adapted solely to freshwater. This species shows the widest longitudinal range of freshwater fish in the world. The burbot is a good model for studies on adaptive genome evolution from marine to freshwater environment. However, no high-quality reference genome has been released. Here, the first chromosome-level genome of the burbot was constructed using PacBio long sequencing and Hi-C technology. A total of 95.24 Gb polished PacBio sequences were generated, and the preliminary genome assembly was 575.83 Mb in size with a contig N50 size of 2.15 Mb. The assembled sequences were anchored to 22 pseudo-chromosomes by using the Hi-C data. The final assembled genome after Hi-C correction was 575.92 Mb, with a contig N50 of 2.01 Mb and a scaffold N50 of 22.10 Mb. A total of 22,067 protein-coding genes were predicted, 94.82% of which were functionally annotated. Phylogenetic analyses indicated that burbot diverged with the Atlantic cod about 44.4 million years ago. In addition, 377 putative genes that appear to be under positive selection in burbot were identified. These positively selected genes might adapt to the freshwater environment. These genome data provide an invaluable resource for the ecological and evolutionary study of the order Gadiformes.