In the last year, new hybrid hop (Humulus lupulus L.) variety Agnus was released for cultivation in the Czech Republic. It has been necessary to prepare the quality system of Agnus identification from other Czech genotypes and characterise the germplasm of this variety by molecular methods. We proved that utilization of five STS primer combinations successfully and completely identified and determined Czech released varieties and new promising breeding materials. The use of STS method was also very effective and sensitive for control of authenticity and purity of variety Agnus in multipropagation cycle. The study of genetic diversity of 61 hop varieties by RAPD, STS, ISSR and AFLP methods confirmed, that germplasm of variety Agnus has ranked among high-alfa varieties. The results can be successfully used for identification, germplasm management, genetic studies and breeding purposes by breeders, multipropagators and hop growers.