The genetic diversity and genetic relatedness of seven indigenous Czech rabbit breeds included in the National Program of Conservation and Utilization of Genetic Resources was evaluated. The set was completed with a modern hybrid strain Hyplus 19 x 59 for comparison. Sixteen microsatellite loci compiled from published sources were multiplexed by 3 or 4 in order to rationalize the reactions and capillary electrophoresis. High level of diversity was demonstrated in all breeds, as expressed by polymorphism information content of the markers and allelic richness, in spite of the limited populations of most of the traditional breeds. On the other hand, the ubiquitous presence of private alleles and substantial genetic distances among the breeds indicated considerable distinctness of all the breeds at the molecular level. The microsatellite profiles were sufficiently characteristic to allow breed assignment of most individuals. The differences among breeds allowed for building an interpretable similarity dendrogram, although without statistical evidence for the hierarchical structure of the multi-breed population. Clustering at the individual animal level and probabilistic assignment of animals to the newly delimited groups consistently revealed distinct subpopulations in two breeds. The degree of inbreeding as a threat to the conservation program was evaluated with the heterozygosity coefficient and the subpopulation inbreeding coefficient F-IS. While heterozygosity corresponded to the panel of European breeds, it remained lower than the values reported for wild and other domestic populations. In conclusion, the conserved rabbit breeds can serve as a reservoir of genetic variants for future breeding despite small living populations.