European grayling, Thymallus thymallus (Linaeus, 1758) is a freshwater fish from the trout family (Salmonidae). It inhabits fast, clean and cold waters. It’s very sensitive to changes in the environment, pollution or destruction. Due to growing human impact to the environment, it’s listed as vulnerable species (VU) in the Red Book of freshwater fish in Croatia in the Danube basin. Since there are not many studies of grayling in Croatia, the objectives of this study were to determine the relationship population of this species, describe the intraspecies structure, determine the degree of intraspecies and intrapopulation genetic diversity, determine populations of indigenous origin, restocking effects and identify particularly important populations for protection in Croatia. Phylogenetic reconstruction was made based on mitochrondrial gene sequence for cytochrome b was performed using the maximum parsimony, maximum likelihood and median joining method. Using the DnaSP program, I determined measures of gene polymorphism, migration rates and effective size between populations were determined using the Migrate program. The results of phylogenetic reconstruction showed a weak structure of populations in Croatia. Low genetic diversity and small effective population size were found. Populations of autochthonous origins located in Slunjčica have been determined. Important populations for protection located in Kupa and Slunjčica have been identified.