The ground squirrel (Spermophilus citellus) is an endemic species of grassy, steppe and pasture habitats in Central and South-Eastern Europe threatened by habitat loss. Rescue programs can mitigate the negative effects of population fragmentation. In this work we evaluated the historical and current distribution range of ground squirrel in Slovakia on the basis of published map data since 1970. Using genomics methods, we analyzed the genetic structure of 18 colonies in Slovakia, in which sveral conservation activities were carried out in recent decades. Using discriminant analysis of principal components (DAPC), we assigned individuals to genetic groups and provided recommendations for controlled translocations to mitigate disrupted gene flow across the fragmented landscape. In Slovakia we have identified a rapid decrease in the number of colonies since 1970. The species distribution data show that the largest population decline occurred after 1995. From a genetic point of view, we identified the studied population of ground squirrels as significantly structured due to the lack of gene flow between colonies. We confirmed an identical genetic structure between the source and translocated colonies, which may indicate a sufficient number of founding individuals and minimal negative consequences of the founder effect. We recommend active conservation actions focusing to improve the quality of habitats and establish the targeted gene flow between colonies within genetic groups.