The mechanism of osmoregulation is crucial for maintaining growth, development, and life activities in teleosts. Takifugu obscurus, the only euryhaline species in the genus Takifugu, is a proper model organism for studying the mechanism of lowsalt tolerance and hypoosmotic regulation. In this study, whole-genome sequencing data were obtained from 90 pufferfish representing five species within this genus, T. rubripes, T. obscurus, T. flavidus, T. niphobles, and T. bimaculatus. Using a phylogeny, PCA, and population structure analyses, we observed similar amounts of population genetic differentiation among species. The five species are closely related to each other and have differentiated within a relatively short period, while T. bimaculatus and T. flavidus shared the most similar genetic backgrounds. We further identified hundreds of genes under selection related to hypoosmotic regulation between T. obscurus and other Takifugu species, including 16 representative genes involving ion transporters (atp1a3, atp2a2, atp2a3, slc13a1, slc5a8, slc12a2, slc12a4, slc26a2, scn1b, and kcna2/3/10), genes involved in hormone regulation (fyn, prlr, and grb2), and a gene associated with water absorption (aqp3). Our findings provide preliminary insight into the mechanism of osmoregulation and will facilitate follow-up validation of candidate genes related to osmoregulation in T. obscurus. Abbreviations SNP Single nucleotide polymorphism PCA Principal component analysis NKCC2 Na+-K+-Cl− cotransporter 2 NCC Na+-Cl− cotransporter NKA Na+/K+-ATPase LD Linkage disequilibrium GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes Responsible Editor: T. Reusch. Reviewed by M. Heckwolf and undisclosed experts.