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Largemouth bass (LMB; Micropterus salmoides) has been an economically important fish in North America, Europe, and China. This study obtained a chromosome-level genome assembly of LMB using PacBio and Hi-C sequencing. The final assembled genome is 964 Mb, with contig N50 and scaffold N50 values of 1.23 Mb and 36.48 Mb, respectively. Combining with RNA sequencing data, we annotated a total of 23,701 genes. Chromosomal assembly and syntenic analysis proved that, unlike most Perciformes with the popular haploid chromosome number of 24, LMB has only 23 chromosomes (Chr), among which the Chr1 seems to be resulted from a chromosomal fusion event. LMB is phylogenetically closely related to European seabass and spotted seabass, diverging 64.1 million years ago (mya) from the two seabass species. Eight gene families comprising 294 genes associated with ionic regulation were identified through positive selection, transcriptome and genome comparisons. These genes involved in iron facilitated diffusion (such as claudin, aquaporins, sodium channel protein and so on) and others related to ion active transport (such as sodium/potassium-transporting ATPase and sodium/calcium exchanger). The claudin gene family, which is critical for regulating cell tight junctions and osmotic homeostasis, showed a significant expansion in LMB with 27 family members and 68 copies for salinity adaptation. In summary, we reported the first high-quality LMB genome, and provided insights into the molecular mechanisms of LMB adaptation to fresh and brackish water. The chromosome-level LMB genome will also be a valuable genomic resource for in-depth biological and evolutionary studies, germplasm conservation and genetic breeding of LMB., (© 2020 John Wiley & Sons Ltd.)