Availability of a contiguous chromosome-level genome assembly is the foundational step to develop genome-based studies in American mink (Neogale vison). The main objective of this study was to provide a high quality chromosome-level genome assembly for American mink. An initial draft of the genome assembly was generated using 2,884,047 PacBio long reads. Integration of Hi-C data into the initial draft led to an assembly with 183 scaffolds and scaffold N50 of 220 Mb. This gap-free genome assembly of American mink (ASM_NN_V1) had a length of 2.68 Gb in which about 98.6% of the whole genome was covered by 15 chromosomes. In total, 25,377 genes were predicted across the American mink genome using the NCBI Eukaryotic Genome Annotation Pipeline. In addition, gene orthology, demographic history, synteny blocks, and phylogenetic relationships were studied in connection with the genomes of other related Carnivora. Furthermore, population-based statistics of 100 sequenced mink were presented using the newly assembled genome. Remarkable improvements were observed in genome contiguity, the number of scaffolds, and annotation compared to the first draft of mink genome assembly (NNQGG.v01). This high-quality genome assembly will support the development of efficient breeding strategies as well as conservation programs for American mink.