Mitochondrial DNA (mtDNA) is maternally inherited and controls the oxygen-related production of adenosine-5'-triphosphate, which is transported from the mitochondria to other cellular compartments and used as energy for cellular activities. The mtDNA is physically separated from nuclear DNA (nDNA). Ionizing radiation (IR) causes the release of both mtDNA and nDNA into circulation. Our previous study demonstrated that nDNA has potential to be a biodosimeter. In this study, branched DNA technology was used to explore the alteration pattern of mtDNA after IR. C57BL/6 mice were exposed to 0, 1.5, 3, 6, 8, or 10 Gy total body irradiation; thereafter, plasma mtDNA was assessed with samples collected at 3, 6, 9, 15, 24, 48, 72, or 168 h. We found that: (1) the designed probesets were specific for mtDNA extracted from the liver, and they recognized the small amount of mtDNA mixed in the nDNA; (2) plasma mtDNA exhibited a statistically significant increase only at 6 h after 8 Gy irradiation. The alteration of mtDNA was not dose-dependent or time-dependent; hence, it is unlikely to be an effective biodosimeter.