Whole-genome duplication and genome compaction have been thought to play important roles in teleost fish evolution. Ayu (or sweetfish), Plecoglossus altivelis, which belong to order Osmeriformes, have a 1-year lifespan and an XX/XY sex-determination system. We produced a draft genome sequence of ayu by whole-genome shotgun sequencing and linkage maps using a genotyping-by-sequencing approach. We further identified sex-associated loci by genome-wide association study, genotyping-by-sequencing, and whole-genome resequencing using wild populations.