Sweet cherry is a vegetatively propagated, perennial plant with high level of heterozygosity and ancient breeding history. Therefore, it is necessary to keep, conserve and evaluate known genetic resources for future breeding programs and fruit production stability. In present, the utilization of DNA molecular genetic analyses is the best suitable method for evaluation of individual accessions, thus we eliminated duplications and characterized the genetic relationships. In our work, we used PCR primer combinations for 19 SSR and 2 EST-SSR loci for analyses of 123 current, old and local sweet cherry cultivars from Czech genetic resources of Research and Breeding Institute of Pomology in Holovousy. In total, 115 polymorphic fragments were amplified, which we used for hierarchical cluster analysis of genetic variability. The result dendrograms were divided into three main clusters and ten subgroups. Clustering corresponded to genealogical and geobotanical characteristics of individual accessions as breeding history of several known accessions.