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Objective: The ayu or sweetfish Plecoglossus altivelis is ray-finned fish that is widely distributed in East Asia. The genome size of ayu was estimated at approximately 420 Mb. Previously, we reported on ayu draft genome assembly by whole-genome shotgun using Illumina short reads and PacBio long reads; however, the assembly was not to chromosome level. Therefore, to improve the draft genome sequence of ayu to chromosome level, we performed in situ Hi-C sequencing as a source of linkage information. Results: The ayu genome assembly yielded 28 large scaffolds that corresponded to the karyotype of ayu (n = 28). The resulting ayu genome assembly has a N50 scaffold length of 17.0 Mb, improved from 4.3 Mb. The high-quality reference genome will be helpful for phylogenetic research on bony fishes and for breeding programs in ayu aquaculture. [ABSTRACT FROM AUTHOR]