The genetic diversity, population structure and gene flow of the Great Bustards (Otis tarda) living in Austria-Slovakia-West Hungary (West-Pannonian region), one of the few populations of this globally threatened species that survives across the Palaearctic, has been assessed for the first time in this study. Fourteen recently developed microsatellite loci identified one single population in the study area, with high values of genetic diversity and gene flow between two different genetic subunits. One of these subunits (Heideboden) was recognized as a priority for conservation, as it could be crucial to maintain connectivity with the central Hungarian population and thus contribute to keeping contemporary genetic diversity. Current conservation efforts have been successful in saving this threatened population from extinction two decades ago, and should continue to guarantee its future survival.