European huchen (Hucho hucho) is a representative of large and rare migratory salmonid fish, which has become endangered due to extensive anthropogenic changes in freshwater ecosystems. Numerous broodstocks of the European huchen have therefore been established throughout the species’ range in recent years to supplement wild fisheries of this species. Unfortunately, this conservation management strategy entails a number of potential ecological and genetic risks associated with the release of farm-raised fish into wild populations. A comprehensive and feasible genetic monitoring protocol for broodstocks maintained for the production of restocking material is therefore essential in the sustainable management of critically endangered fish species. The current paper presents phylogenetic characteristics of four selected huchen broodstocks across Central and Eastern Europe. Genetic comparisons of the studied broodstocks were based on ten microsatellite DNA markers. The effective population size (Ne), the individual assignment test, the Principal Coordinates Analysis (PCoA), the allele sharing distance (DAS) and the Bayesian clustering analysis were applied in this study. Moreover, five selected fragments of mitochondrial DNA were used for molecular verification of species membership and genetic purity of examined specimens.