The multidisciplinary approach is in increasing use in modern science for solving complicated problems. Molecular genetics not only helps us understand biological processes, such as evolution and speciation, but also sheds light on numerous historical questions as to the directions of peoples’ migrations, the degree of interpenetration of contemporaneous cultures, and their continuity. In particular, investigation of phylogenetic relationships of domestic animals allows us to detail the interactions between bearers of different archaeological cultures. In the Bronze Age, Inner Asia and adjacent territories were characterized by intense human migrations and rapid spread of productive economies, including livestock farming. Here we examine the phylogenetic patterns of horses from two important Bronze Age Ob-Irtysh cultures in Western Siberia, the Sargarinsko-Alexeevskaya and Irmen ones, and the degree of their genetic proximity to horses from earlier (Andronovo and Eluninskaya) and later (Khereksur and “Deer Stone”, Biykenskaya, Bystryanskaya and Pazyryk) cultures in the region and adjacent territories. Data obtained from sequencing and analysis of mitochondrial genomes reveal differences in the mitochondrial gene pool of horses from the Sargarinsko-Alexeevskaya and Irmen cultures of the south of Western Siberia, highlighting the unique mitochondrial genetic diversity of the original horse herds of these cultures and the lack of close breeding contacts between them. We demonstrate an overlap between the mitochondrial gene pools of horses from the Khereksur and “Deer Stone” cultures of Mongolia and the Andronovo culture. We also established continuity between many of the obtained haplotypes of horses from the Early, Developed, Late Bronze Age, and Early Iron Age in southern Western Siberia, indicating the preservation of a significant part of the maternal gene pool diversity of domestic horses in the region across several historical and cultural periods. The similarity of mitochondrial haplotypes among horses of the Sargarinsko-Alexeevskaya culture, modern horses of the Akhal-Teke breed of Central Asia, and indigenous breeds of East Asia and Southern Europe, as well as between horses of the Irmen culture and modern horses of local breeds in Northern Europe, may reflect the migration routes of bearers of these cultures after their disintegration in the region under study. It also characterizes features of the formation of these breeds in ancient times. However, nuclear genetic markers should also be investigated to corroborate these hypotheses.