Mitochondrial DNA in Tumor Evolution and Heterogeneity: Characteristics, Technological Advances, Applications and Challenges

Tumor heterogeneity and Darwinian evolution act as core drivers of therapeutic resistance. Deciphering the phylogenetic architecture of cancer demands high‐resolution lineage tracing. Nevertheless, exogenously introduced lineage markers possess inherent limitations. This review presents somatic mitochondrial DNA (mtDNA) mutations as a powerful, newly emerging endogenous lineage marker. It describes the unique characteristics of mtDNA, including high copy number, elevated mutation rate, and maternal inheritance modulated by random genetic drift, which support its function as a natural, high‐resolution cellular barcode. It then summarizes critical technological breakthroughs for mtDNA mutation detection, particularly under single cell and spatial multi‐omics frameworks. The central section integrates pioneering phylogenetic tree applications to clarify metastatic patterns, dissect resistance evolution, and correlate clonal history with phenotypic states. It also addresses ongoing methodological and interpretive obstacles, such as nuclear mitochondrial DNA segments (NUMTs) and functional consequences of mutations. Future directions include improving detection sensitivity, adopting long‐read sequencing, and translating mtDNA lineage data into clinical strategies for minimal residual disease monitoring and adaptive therapy guidance. In summary, lineage tracing based on mtDNA mutations has become an indispensable tool, offering unprecedented insights into tumor biology and facilitating precision oncology advancement.