Four broodstocks of European huchen (Hucho hucho) from Poland, Germany, Slovakia, and Ukraine were investigated using five selected fragments of mtDNA. The mitochondrial DNA sequence analysis was successfully applied to the Polish and German broodstocks of European huchen for the first time. A very low haplotype (h = 0.097) and nucleotide (π = 0.00013) diversity across 3573 bases of mtDNA fragments (partial regions of NADH-1, ATPase 6, Cytochrome b, evidenced by three closely related mtDNA haplotypes were found. The analysis of pairwise genetic differentiation (Fst) displayed a statistically significant differentiation between German (clade A) and the rest of examined broodstocks (clade B), clustering them into two separate groups. Moreover, the applied mtDNA markers did not reveal any differences among fish from the clade B, suggesting that other markers should be used to display a deeper genetic background of the studied broodstocks. The two clusters of European huchen distinguished under the current study should be considered as distinct management units by managers, who may be tempted to transport brood fish or yearlings across the range of European huchen distribution. It means that stocking material and spawners of European huchen from the upper parts of the Danube River (Clade A) should not be mixed with those from the lower parts (Clade B). Moreover, for any supplementation of broodstocks in order to increase their genetic variability only the fishes within the described management units should be used.