European beech (Fagus sylvatica L.) is one of the most important broadleaved tree species in Europe both ecologically and economically. Nowadays, in the Czech Republic, beech is underrepresented in forest tree species composition, and there are tendencies to increase its proportion. When reintroducing beech, genetic variability, along with other factors, play a key role. The main aim of this study was to evaluate the genetic diversity of ten selected indigenous beech populations across the Czech Republic. Two hundred and fifty individuals were genotyped on 21 polymorphic nuclear microsatellite markers, which were amplified using two newly assembled multiplexes. According to the results, observed heterozygosity (Ho) among populations ranged from 0.595 to 0.654 and expected heterozygosity (He) from 0.650 to 0.678. That is comparable with the findings in other European studies. The high discriminatory power of the assembled multiplexes was confirmed by calculating the Probability of Identity among both unrelated and related individuals. Principal Coordinate Analysis (PCoA) based on Nei's genetic distances revealed that there are genetic differences among populations resulting in three approximate clusters (geographically north, south-east, and south-west). Nevertheless, the results implicate that on a geographical scale of the Czech Republic, the distance is unlikely to be the primary driver of genetic differentiation.