Abstract The male-specific DNA markers are very useful in molecular sexing of non-flowering plants and seeds of dioecious species. In this paper we identified ten Y chromosome-specific RAPD primers suitable for identification of male plants in three Cannabaceae species with sex chromosomes (Humulus lupulus, XX/XY; H. japonicus, XX/XY1Y2; Cannabis sativa, XX/XY). Basing on the nucleotide sequence of the OPJ-09 RAPD product we developed the HJY09 SCAR marker, which is very efficient in sexing of Japanese hop.