Multiple plant lineages have independently evolved sex chromosomes and variable karyotypes to maintain their sessile lifestyles through constant biological innovation.Morus notabilis, a dioeciousmulberryspecies, has the fewest chromosomes amongMorusspp., but the genetic basis ofsex determinationandkaryotype evolutionin this species has not been identified. In this study, three high-quality genome assemblies were generated forMorusspp. [including dioeciousM.notabilis(male and female) andMorus yunnanensis(female)] with genome sizes of 301–329 Mb and were grouped into six pseudochromosomes. Using a combination of genomic approaches, we found that the putative ancestral karyotype ofMorusspecies was close to 14 protochromosomes, and that several chromosome fusion events resulted in descending dysploidy (2n= 2x= 12). We also characterized a ∼ 6.2-Mb sex-determining region on chromosome 3. Four potential male-specific genes, a partially duplicated DNA helicase gene (namedMSDH) and threeTy3_Gypsylong terminal repeat retrotransposons (namedMSTG1/2/3), were identified in the Y-linked area and considered to be strong candidate genes for sex determination or differentiation. Population genomic analysis showed that Guangdong accessions in China were genetically similar to Japanese accessions of mulberry. In addition, genomic areas containing selective sweeps that distinguish domesticated mulberry from wild populations in terms of flowering and disease resistance were identified. Our study provides an important genetic resource for sex identification research and molecular breeding in mulberry.