The European ground squirrel (Spermophilus citellus) is considered an endangered species with declining numbers throughout Europe, most pronounced at the western margin of its distribution area. Being extinct in Germany and Poland, the western margin of its distribution is in the Czech Republic. Here, landscape fragmentation has restricted the ground squirrels into few and very isolated localities where local extinctions still occur. In the present study we analysed European ground squirrels from six Czech and one Slovak localities using five microsatellite loci as genetic marker. The results show a strong genetic differentiation among the investigated populations (mean value of FST = 0.16) and high levels of inbreeding (values of FIS ranged from 0.34 to 0.90). High level of inbreeding is generally considered to affect the viability of each population, which could lead to extinction. One of the most important factors is the lack of migration due to the large distances between the populations and the presence of migration barriers. Based on the results obtained we recommend a few suggestions for a conservation management of this species.