The main problem in biodiversity conservation is a loss of genetic diversity in structured populations, which is often consequence of habitat contractions and a limited gene flow. These dynamic biological processes require genetic monitoring, environmental and landscape data. Translocations represent an important conservation tool to temper the negative genetic consequences of the landscape fragmentation and habitat loss. European ground squirrel is the suitable model species to study the consequence of fragmentation of the landscape also in habitat specialist species of forest ecosystem, considering to his quick population turnover, specific habitat´s demand and relatively simple techniques of monitoring and capturing. On the other hand, forest habitat specialists live in harsh conditions, are hardly to be observed and genetically sampled for genomic analysis. We studied the consequences of habitat´s fragmentation to genetic diversity of European ground squirrel in selected colonies in Western Carpathians. Genomic DNA was extracted from tail cuts and extracted DNA was analysed using ddRAd sequencing protocol (double digested restriction site associated dimorphism). After cleaning and filtering of sequences we identified 6,904 loci. Subsequently, we performed population genetic analyses for evaluating the parameters of the genetic diversity of individual colonies. We found that the geographically isolated colonies of ground squirrel living in the areas of the Detva, Heľpa and Horeša willage, showed the lowest parameters of genetic diversity from a set of 18 analysed colonies. We assume that these colonies are long-term isolated and due to small effective population size, there are ongoing negative processes of genetic drift causing the loss of genetic diversity. Loss of genetic diversity can cause disability of colonies to respond to environmental changes and natural disasters. It can lead them to extinction. In these isolated colonies, we emphasize the need for active conservation actions including augmentations of colonies by translocation of genetically diverse individuals.