Maternal transmission of mitochondrial DNA (mtDNA) allows us to generate mtDNA congenic strain by repeating backcrosses of female mice to male mice of an inbred strain, which carries different mtDNA haplotype from that of the female progenitor. Since genetic backgrounds of inbred strains commonly used (e.g., C57BL/6J [B6] and BALB/c) are mainly derived from an European subspecies of Mus musculus domesticus, congenic strains, in which mtDNA originated from an Asian subspecies M. musculus musculus or an European species M. spretus, give in vivo condition that mismatch occurs between the mitochondrial and the nuclear genome. So far, little has been known how the mismatch condition affects the physiological phenotype of the mice. To address this question, we established two mtDNA congenic strains, C57BL/6J(B6)-mtSPR and BALB/c-mtSHH, which carry M. spretus- and M. m. musculus-derived mtDNAs, representing the conditions of interspecific and intersubspecific mitochondrial-nuclear genome mismatch, respectively. Using these congenic strains, we examined their physical performance by measuring their running time on a treadmill belt until exhaustion. The result clearly showed that the mtDNA congenic strains manifested a significant decrease in the level of physical performance, when compared with their progenitor strains. It also appeared that the congenic mice manifested growth rate. Thus, all results indicated that mismatch between the mitochondrial and the nuclear genome causes phenotypic changes in individuals of mice.