Sexuality of crops affects both cultivation and breeding systems. Cultivated persimmon (Diospyros kaki Thunb) has a morphologically well-characterized polygamous or gyonodioecious sexual system. However, the genetic basis of sexuality in D. kaki has yet to be characterized. Here, we used dioecious D. lotus L., a diploid wild relative species to hexaploid or nonaploid D. kaki, as a model to clarify the genetic basis of sexuality in Diospyros and to develop molecular markers associated with the sexuality of individuals. Using 62 F-1 offspring segregated into distinct male/female phenotypes, we found two amplified fragment-length polymorphism markers, DlSx-AF4 and DlSx-AF7, which cosegregated with maleness. This could suggest that the sexuality of D. lotus is controlled by a single gene/haploblock, and the male is dominant over the female. Thus, D. lotus's sexuality can be described as the heterogametic male type, the ICY-type, as reported for most other dioecious plant species. For unknown reasons, segregation of the phenotype of a sequence-characterized amplified region marker developed from DlSx-AF4 (DlSx-AF4S) and/or the male/female phenotype in two different crosses in D. lotus showed an apparent bias towards femaleness and better fitted 1:2 than 1:1, which is the theoretical segregation for a single genetic locus or haploblock in diploid D. lotus. DlSx-AF4S could distinguish D. kaki cultivars with female and male flowers from cultivars with only female flowers, strongly indicating that the same genetic system controls D. kaki's sexuality and that DlSx-AF4S could be used as a genetic marker for sexuality in D. kaki breeding programs.