Mitochondrial DNA (mtDNA) deletion mutations are proposed contributors to aging-related muscle fiber loss and atrophy, but evidence of a causal role for these mutations in muscle aging is lacking. Elucidating the etiology of in vivo mtDNA deletion mutations will help to better understand and test the possible roles of these mutations in aging. The implication of mtDNA mutations in aging is based on the susceptibility of mtDNA to oxidative damage by reactive oxygen species (ROS) due to residing in mitochondria, the primary source of endogenous ROS. Cells possess many pathways for neutralizing ROSs, including a variety of superoxide dismutases (SOD). Mice lacking CuZnSOD (Sod1(-/-) mice) have high levels of oxidative damage in many tissues including skeletal muscle and are a model for testing the role of oxidative damage in the formation of mtDNA deletion mutations. The increased DNA oxidative damage in Sod1(-/-) mice is associated with increased mtDNA deletion mutations in a variety of tissues, but skeletal muscle mtDNA mutations have not been reported. We hypothesized that a life-long absence of mouse muscle CuZnSOD would increase mtDNA deletion mutation frequency and focal accumulation of these mutations in aging mouse skeletal muscle. Focal accumulations of mtDNA deletion mutations were detected by histochemical staining for cytochrome c oxidase (cytOX) activity and detection of cytOX-negative fibers, a marker of focal mtDNA mutation accumulation, within approximately 20,000 muscle fibers through a distance of 1000μm. Total DNA was extracted from intervening unstained sections and mtDNA deletion mutation frequency was measured by a droplet digital PCR. Droplet digital PCR quantification of mtDNA deletion mutations showed no difference in mtDNA deletion mutation frequency in Sod1(-/-) mouse muscle compared to wild-type mice and we observed no significant increase in the number of cytOX-negative muscle fibers, in Sod1(-/-) mice compared to wild-type mice. These data demonstrate that not all changes in cellular oxidative stress are linked to mtDNA deletion mutations and shift the focus to other etiologies for these mutations that need to be clarified to better test their possible role in aging.