Hop (Humulus lupulus L.) is economically important dioecious plant in the family Cannabaceae, which has the biggest usefulness in brewing industry. Even though from the breeding point of view male hop plants are extraordinarily important, from the economic point of view male hop plants are unwanted in hop cultivars, because only unfertilized female flowers (hop cones) have an economic value. In breeding early determination of sex in hop seedlings is very important so part of male seedlings can be planted on a yield at another location. Until now determination of seedlings sex has been possible with 100 % reliability only phenotypically, based on morphological differences of generative organs during flowering season. With the same reliability this is now possible in seedling phase using DArT molecular markers. In present M. Sc. Thesis we had successfully introduced and optimized multiplex PCR with five molecular markers, of which four are male-specific, and one that correlates with chloroplast DNA and has no linkage with sex (this one amplifies in all male and all female plants – internal control of PCR amplification). With this method sex of 99.7 % of 295 hop seedlings from 15 different crossing families was successfully determinated. We showed stability and repeatability of the method. We also introduced a protocol for rapid DNA isolation (for the first time in hop plant). With this study we had importantly influenced on hop breeding process, because we had decreased workload and expenses, which are linked with determination of male hop plants.