Abstract Multiple plant lineages have independently evolved sex chromosomes and variable karyotypes to maintain their sessile lifestyles through constant biological innovation. Morus notabilis, a dioecious mulberry species, has the fewest chromosomes among Morus spp., but the genetic basis of sex determination and karyotype evolution in this species has not been identified. In this study, three high-quality genome assemblies were generated for Morus spp. [including dioecious M. notabilis (male and female) and Morus 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 of Morus species 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 (named MSDH) and three Ty3_Gypsy long terminal repeat retrotransposons (named MSTG1/2/3), 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.