Understanding the biology of mitochondrial DNA (mtDNA) at the single-cell level has yielded important insights into inheritance, disease, and normal aging. In nuclear gene disorders of mtDNA maintenance, neurodegeneration, and aging, different somatic mtDNA mutations exist within individual cells and may be missed by techniques applied to whole tissue DNA extract. We therefore provide a method for characterizing mtDNA within single skeletal muscle fibers. During embryogenesis, mtDNA content is subject to a tight bottleneck and this may account for differential segregation of mutant mtDNA in offspring. We also present a method to study this phenomenon by single-cell analysis of embryonic PGCs (primordial germ cells).