Chromosome-level de novo genome assembly and whole-genome resequencing provide insights into genome evolution and sex determination of sleepy cod Oxyeleotris lineolata (Steindachner 1867).

The sleepy cod (Oxyeleotris lineolata Steindachner 1867) is a valuable freshwater teleost with male-biased growth dimorphism, making sex control breeding vital for aquaculture. We generated a high-quality chromosome-level genome assembly (⁓985.07 Mb) using PacBio HiFi and Hi-C sequencing, with 99.31% sequences anchored to 23 chromosomes (contig N50: 14.73 Mb; scaffold N50: 42.32 Mb). BUSCO assessment showed 98.1% completeness, with 25,933 protein-coding genes and 42.21% repetitive elements annotated. Phylogenetic analysis indicated closest affinity to Gobiidae (Boleophthalmus pectinirostris, Periophthalmus magnuspinnatus), divergence ∼59.78 Ma and 301 expanded / 390 contracted gene families. Whole-genome resequencing of 17 females and 16 males identified 274,561 high-confidence SNPs. Integrating heterozygosity analysis and GWAS, we identified a sex-associated region on chromosome 2, with gsdf as the candidate sex-determining gene. A sex-specific SNP supported ZZ/ZW sex-determination system. This study provides key genomic resources for O. lineolata evolution, development and aquaculture optimization, and lays molecular foundation for all-male breeding.