Mitochondrial genomes (mtDNA) have become invaluable in species classification and evolutionary studies due to their unique characteristics, including maternal inheritance, and high mutation rates. This review examines the application of mtDNA in tracing evolutionary history, elucidating phylogenetic relationships, and understanding mechanisms of species divergence. The evolution of mitochondrial DNA research from its initial focus on energy metabolism to its current role in biodiversity assessments highlights its significance in modern biology. Mitochondrial DNA barcoding, particularly utilizing the cytochrome c oxidase I (COI) gene, has revolutionized species identification, enabling rapid and accurate classification across diverse taxa. The article further explores the implications of mtDNA in understanding adaptive evolution, as genetic variations within mitochondrial genomes can reveal insights into how species respond to environmental pressures. However, challenges such as gene mixing, hybridization, and incomplete lineage sorting can complicate interpretations of mtDNA data. Thus, integrating mitochondrial with nuclear genome data is advocated to provide a comprehensive view of species relationships and evolutionary patterns. Future research directions emphasize the need for multi-genome studies, investigations into ecological adaptations, and exploration of understudied taxa and ecosystems, which are crucial for enhancing our understanding of biodiversity and informing conservation strategies.