The high level of diversity of the genetic structure of mitochondrial DNA and inheritance through the maternal line make it a unique object for studying evolutionary processes, phylogenetic relationships and assessing population diversity. 64 thoroughbred horse mares were selected from 20 main dam families and the most promising branches for analysis of the non-coding region of the mtDNA D-loop. Primers for amplification of the mtDNA D-loop region were selected taking into account the reference sequence of the fossil Swedish horse X79547. GeneBank data was used for 17 haplogroups located under the access number (n = 83): JN398377-JN398457, EF597513-EF597514 for identifying the obtained nucleotide sequences. The 530 mtDNA D-loop region analysis was performed using the MEGA4 program. 23 haplotypes corresponding to 8 haplogroups A, B, G, H, I, L, M and N according to the classification of Achilli et al. (2012) were identified in the process of studying the polymorphism of the hypervariable region of the mtDNA D-loop in dam families of thoroughbred horse breed. Among mares from 20 dam families 16 were identified as belonging to only one haplogroup. 2 haplogroups were identified in 4 dam families of this breed (4,5,9, 19) due mainly to transitions at different breeding periods of thoroughbred riding horses. The genetic analysis of the mitochondrial genome of the main dam lines of thoroughbred horse breed indicates a rather high level of diversity of mtDNA haplotypes and a clear differentiation of female families in the domestic population of this breed..