Somatic mitochondrial DNA (mtDNA) mutations are frequently observed in tumors, yet their role in pediatric cancers remains poorly understood. The heteroplasmic nature of mtDNA-where mutant and wild-type mtDNA coexist-complicates efforts to define its contribution to disease progression. In this study, bulk whole-genome sequencing of 637 matched tumor-normal samples from the Pediatric Cancer Genome Project revealed an enrichment of functionally impactful mtDNA variants in specific pediatric leukemia subtypes. Collectively, the results from single-cell sequencing of five diagnostic leukemia samples demonstrated that somatic mtDNA mutations can arise early in leukemogenesis and undergo positive selection during disease progression, achieving intermediate heteroplasmy-a "sweet spot" that balances mitochondrial dysfunction with cellular fitness. Network-based systems biology analyses link specific heteroplasmic mtDNA mutations to metabolic reprogramming and therapy resistance. We reveal somatic mtDNA mutations as a potential source of functional heterogeneity and cellular diversity among leukemic cells, influencing their fitness and shaping disease progression.