High-oleic peanuts are increasingly valued in agricultural production and consumer markets. Nevertheless, limited genomic information hinders the integration of genetic analyses and modern breeding strategies. This study details a chromosome-level genome assembly of Kaixuan 016, a high-oleic peanut variety developed through gamma-radiation-assisted breeding, exhibiting enhanced agronomic traits. Utilizing PacBio long-read sequencing and Hi-C technology, the assembly encompasses 2532.9 Mb, with a contig N50 of 11.48 Mb, structured into 20 pseudochromosomes. Repetitive elements comprise 72.26 % of the genome. Of the 69,845 protein-coding genes, 97 % are annotated in public databases. Phylogenetic analysis reveals divergence times of approximately 0.3 million years ago (Mya) for the A subgenome and 0.2 Mya for the B subgenome, with their initial divergence occurring around 2.6 Mya. Additionally, our study includes enrichment analyses on species-specific genes, along with expanded, contracted, and positively selected gene families engaged in vital metabolic and biosynthetic pathways such as photosynthesis, linoleic acid metabolism, and fatty acid elongation-key processes for oleic acid accumulation in developing seeds. Comparative genomic analysis with the AT1-1 parent genome suggests that γ-radiation primarily induces structural variations in Kaixuan 016. Population analysis further differentiated Kaixuan 016 and its derivatives into two distinct clusters-Kainong and Jihua accessions, highlighting their superior agronomic traits. This high-quality genome assembly provides a vital resource for comparative genomic studies and facilitates the advancement of accelerated, genome-guided breeding efforts in high-oleic peanuts.