Genotype structure of Acipenser ruthenus was investigated using microsatellites and mtDNA sequence analyses of 247 specimens of 13 locations from the rivers Danube, Volga and Kuban. These studies were done as an essential prerequisite for the conservation of the uppermost relict sterlet population which is located in the Danube River near the border between Germany and Austria. In contrast to the Volga population, which shows weak signs of a separation in at least three sub-populations, both microsatellite profiles and mitochondrial sequences indicate a panmictic population of Danube sterlets. Importantly, introgression of non-native Volga sterlets and a few hybrids with Acipenser baerii were discovered. We observed approximately 23% of non-native sterlets with complete or at least partly Volga background within the Danube sterlets. Nevertheless, approximately 77% of Danube sterlets were pure native fish. We would like to suggest using these natives for supportive breeding and stocking to avoid any possible outbreeding depressions.