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Since one of the peculiarities of the evolutionary history of representatives of the genus Equus (horses, asses and zebras) is the high similarity of the morphology of the skeletons of different species of this genus, morphometric characteristics do not always make it possible to determine the species identity of the discovered horse bone samples. This is especially true for scattered bones and incomplete teeth found separately from whole skeletons. In such a situation, molecular genetic analysis becomes important, which makes it possible to more accurately determine the species. On the basis of mitogenomic data alone, it is difficult to determine whether a sample belongs to a domestic or wild horse; this requires genome-wide analysis. Based on the mitogenome data, it is possible to distinguish clearly different species of the genus Equus from each other. In this work, the authors studied 4 bone samples of ancient horses and 1 bone sample of a medieval horse, found in the Middle Volga region and Southern Siberia. Using the target enrichment method, mitogenomic libraries were obtained for them and their high-throughput sequencing was carried out. Secondary bioinformatics analysis and subsequent analysis of individual reads and phylogenetic reconstructions based on the consensus sequences of the mitogenomes of the studied samples, modern horses from different regions of the world and reference sequences of the domestic horse, Lena horse, Ovodov horse and onager demonstrated the attribution of the studied samples to the above-mentioned species of the genus Equus. Authors’ data showed the preference for using genome-wide sequences to determine the species identity of ancient and modern samples compared to the analysis of individual genetic markers.