Xiongnu was a confederation of nomadic pastoral tribes (~200 bc–100 ad) that founded the first nomadic empire in Central Asia. According to archeological and historical data, the tribes played a key role in ethnic and cultural processes in Central Asia and adjacent regions of Eurasia. Genetic studies of the Xiongnu published to date have focused on remains from burial grounds in present-day Mongolia, in the southern part of the ancient Xiongnu area. However, paleoanthropological materials from numerous Xiongnu cemeteries and settlements in Transbaikalia (the southern region of Eastern Siberia, Russia) in the northern part of the Xiongnu Empire have not been examined genetically. Here, we analyzed mitochondrial DNA variation in a Transbaikalian Xiongnu population based on ancient DNA obtained from skeletal remains (n = 18) at four burial grounds to complement available Xiongnu genetic diversity data. We detected 16 mitochondrial DNA haplotypes belonging to seven East Eurasian haplogroups (A, B5, C, D4, G2a, N9a, and Y) in the Transbaikalian Xiongnu series. We observed substantial similarity between Transbaikalian and Mongolian Xiongnu series with respect to main haplogroup composition and frequencies. We observed several mitochondrial DNA clusters (N9a, Y, B5, and A16) and 11 of 16 haplotypes that were previously undetected in the Xiongnu gene pool. We also observed high similarity between the Xiongnu and contemporary indigenous populations of eastern Central Asia, particularly Mongolian-speaking groups. These findings extend our knowledge of Xiongnu genetic diversity.