Simple SummaryLocal, autochthonous sheep breeds are an important part of the socio-economic and production system in Bulgaria and Europe. Molecular characterization is a key factor for their preservation. In this study, we analyzed the genetic diversity and population structure of Bulgarian autochthonous sheep breeds using a set of 13 microsatellite markers. A total of 600 individuals from 50 flocks representing 12 breeds were included in the study. Our results showed that two of the breeds—Local Stara Zagora/SZ/ and Local Karnobat/MK/—were homogenous in terms of their genetic structure and could be easily differentiated from each other and from all other breeds. At the same time, all of the remaining breeds were an admixture with a heterogenous genetic structure, with some exceptions where occasional flocks were relatively homogenous within some of the breeds. Our study showed that it is necessary to implement proper management practices and effective sheep breeding strategies in Bulgaria to preserve the autochthonous breeds, maintain the genetic diversity, and prevent the erosion of the breed purity. The applied set of microsatellite markers could be further used as an effective molecular tool for the monitoring and development of such programs and strategies.AbstractThis study attempts to provide a deeper insight into the current genetic status of 12 Bulgarian autochthonous sheep breeds using microsatellite (SSR) markers. A total of 600 individuals from 50 flocks were analyzed using a panel of 13 SSR markers. In total, 228 alleles were found in the studied microsatellite loci. The mean number of alleles, the effective number of alleles, and the polymorphic information content (PIC) values per locus were 17.54, 5.250, and 0.799, respectively. The expected heterozygosity (He) for all breeds ranged from 0.70 to 0.82. The within-population heterozygote deficit (Fis) varied from −0.03 to 0.1, reflecting significant levels for 10 of the 12 breeds. The average genetic differentiation (Fst) was 0.046, revealing a low discrimination between the breeds. The genetic distance, principal coordinate analysis, and the structure analysis showed that two of the studied breeds—Local Stara Zagora/SZ/ and Local Karnobat/MK/—were the most distinct sheep populations. The Bayesian clustering approach suggested poor breed differentiation for the remaining 10 sheep breeds. The results suggest that proper management strategies and specific breeding policies need to be implemented in Bulgaria to avoid the intermixing of breeds and to reduce the erosion of breed purity observed in some breeds.