In this study genetic diversity, population structure and genetic relationships of Tsigai populations in Slovakia were investigated using microsatellite markers. Altogether 195 animals from 12 populations were genotyped for 16 microsatellites. 212 alleles were detected on the loci. The number of identified alleles per locus ranged from 11 to 35. In the majority of the populations heterozygosity deficiency and potential risks of inbreeding could be determined. High values of FST (0.133) across all the loci revealed a substantial degree of population differentiation. The estimation of genetic distance value showed that the Slovak Vojin population was the most different from the other populations. The 12 examined populations were able to group into 4 clusters. With this result our aim is to help the Slovak sheep breeders to establish their own mating system, to avoid genetic loss and to prevent diversity of Tsigai breed in Slovakia.