Pufferfish are ideal models for vertebrate chromosome evolution studies. The yellowbelly pufferfish, Takifugu flavidus, is an important marine fish species in the aquaculture industry and ecology of East Asia. The chromosome assembly of the species could facilitate the study of chromosome evolution and functional gene mapping. To this end, 44, 27 and 50 Gb reads were generated for genome assembly using Illumina, PacBio and Hi-C sequencing technologies, respectively. More than 13 Gb full-length transcripts were sequenced on the PacBio platform. A 366 Mb genome was obtained with the contig of 4.4 Mb and scaffold N50 length of 15.7 Mb. 266 contigs were reliably assembled into 22 chromosomes, representing 95.9% of the total genome. A total of 29,416 protein-coding genes were predicted and 28,071 genes were functionally annotated. More than 97.7% of the BUSCO genes were successfully detected in the genome. The genome resource in this work will be used for the conservation and population genetics of the yellowbelly pufferfish, as well as in vertebrate chromosome evolution studies.