Cultivated Diospyros kaki, otherwise known as persimmon or kaki, is a polygamous species, whereas most other Diospyros species are dioecious. Most commercial cultivars of D. kaki bear only female flowers; however, some cultivars bear male flowers as well. Elucidation of the genetic and molecular basis of sex expression in D. kaki may lead to the development of artificial control of sex expression in this important fruit species. To this end, Diospyros lotus was used as a model to study the sexuality of D. kaki. Using a population of 63 F1 offspring of D. lotus, a bulked segregant analysis with amplified fragment length polymorphisms (AFLP) showed that the sexuality of this species is controlled by a single locus or haploblock in a heterogametic male (XY) system. Male-specific subsequences from Illumina HiSeq genomic reads data obtained from male and female pools were cataloged and assembled. This enabled comprehensive isolation of the male-determining regions on the Y-chromosome of D. lotus. Integration of transcriptome and evolutionary analyses led to the identification of one Y-specific sex-determinant candidate gene, OGI. This gene encodes a small RNA that triggers a transitive RNAi on the feminizing gene, MeGI, which is located on the autosome or the pseudoautosomal region of the sex chromosome. Molecular markers developed from the OGI sequence and its flanking regions indicated that the genetic control of maleness expression in D. kaki is based essentially on the OGI/MeGI system.