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The aim of present study was to assess the level of genetic diversity within three autochthonous chicken breeds, as well as to identify the genetic relationships between them based on microsatellite data. In this study, overall 78 animals belonging to the yellow-brownish Oravka (Ob), the white Oravka (Ow) and the Sumavanka (S) breeds were included. The genomic DNA was extracted from feather samples by using commercial kit according to Wizard® Genomic DNA Purification Kit Protocol (Promega Corporation). A panel of eight microsatellite markers (Lei166, Lei192, Lei228, Lei229, Lei234, Lei254, MCW34, and MCW69) were used for animals' genotyping. The level of genetic diversity within each breed was determined based on the calculation of mean number of alleles (MNA), observed heterozygosity (Ho), gene diversity expressed as expected heterozygosity (He), effective allele number (Na) and Shannon's information index (I) using Genalex v6.1. The amount of inbreeding-like effect within breeds, expressed by the Wright's FIS index, was calculated using FSTAT v2.9.3.2. The genetic relationships among breeds were estimated based on the Nei's genetic distance and Wright's FST index. In addition, the discriminant analysis of principal components (DAPC) was used to examine the genetic structure of breeds under consideration. The mean number of alleles across breeds ranged from 2.50 ± 0.33 (Ow) to 8.00 ± 1.43 (Ob), which signalized a certain decrease of genetic variability mainly in case of the White Oravka and the Sumavanka breeds. This decrease also confirmed the obtained values of effective number of alleles (2.13 to 3.46) and Shannon's information index (0.75 to 1.36). Similarly, the gene diversity indicated higher proportion of heterozygosity within the yellow-brownish Oravka (0.63 ± 0.08) compared to the white Oravka (0.46 ± 0.08) and Sumavanka (0.44 ± 0.10) breeds. However, despite the higher proportion of heterozygotes within the yellow-brownish Oravka, the Wright's FIS index showed that this population can be affected by a relatively strong impact of inbreeding (FIS = 0.17). The lowest value of FIS index (0.03) pointing to low effect of relatives mating was found in the Sumavanka breed (FIS = 0.03). The average values of Nei's distance (0.93 ± 0.23) and FST index (0.33 ± 0.08) showed that the populations were genetically differentiated. As expected, due to the genetic background of analysed breeds, the highest genetic similarity was found between the white and yellow-brownish Oravka, while the highest genetic distance showed the white Oravka and the Sumavanka breeds. Moreover, the DAPC analysis indicated that between the white and yellow-brownish Oravka some level of admixture can be found. The obtained three discriminant functions corresponded to 67.4% of total genetic variance conserved in the analysed dataset. Based on the study results it can be concluded that the state of genetic diversity within analysed autochthonous chicken breeds should be monitored constantly. Moreover, the increase in a sample size could be beneficial to describe their gene pool in a more detailed level, as well as to identify genetically most important individuals for the sustainable management of animal genetic resources in future. [ABSTRACT FROM AUTHOR]