In this study, the intra- and inter-population level of genetic diversity of 26 transboundary and local sheep breeds reared in the Czech Republic was analysed. A total of 14,999 animals genotyped for 11 microsatellite markers were included to describe the gene pool of the breeds. The level of genetic diversity was derived from the proportion of heterozygous animals among and within breeds. The average polymorphic information content (0.745) and Shannon’s index (1.361) showed a high genetic variability of the applied set of genetic markers. The average observed heterozygosity (0.683 ± 0.009), as well as FIS index (-0.025 ± 0.004), pointed to a sufficient proportion of heterozygotes concerning the loss of genetic diversity. The deficit of heterozygotes was most evident in Cameroon sheep (FIS = 0.036). The Nei’s genetic distances and Wright’s FST indexes showed that the analysed breeds are genetically differentiated to separate clusters with Cameroon sheep as the most genetically distant breed. Individual variation accounted for 83.2 % of total diversity conserved across breeds, whereas 16.8 % of genetic similarity resulted from the inter-population reduction in heterozygosity.