Cannabis sativa is a mainly dioecious plant, which means that female and male flowers develop on separate individuals, which is controlled by an XY sex determination system: females have two X chromosomes and male plants carry an X and a Y chromosome. C. sativa is a crop that has a wide variety of applications, some of which depend on the sex of the plant. Females are, for example, used for the cannabinoid production, as cannabinoids are produced in the female inflorescence. However, while adult C. sativa individuals present high sexual dimorphism, it is not possible to phenotypically distinguish male and female plants before flowering. Here we present the identification of a highly conserved sex marker, CsPDS5, and the development of a robust, reliable and affordable PCR-based method to determine the sex genotype. In contrast to other sex genotyping methods, our approach relies on a gene polymorphic between the X and Y chromosomes and therefore requires only a single PCR with one pair of primers. The method was tested in 14 hemp-type cultivars and 6 crosses, with different tissues and developmental stages, on more than 500 samples, with 100% accuracy. Our assay allows early sex identification and hemp selection, which is useful for both research and industrial purposes. Finally, the pipeline presented here to identify genes polymorphic between the X and Y chromosomes can serve to discover new sex markers, not only in C. sativa but also in other dioecious plants and other organisms with 2 sexes.