Abstract: Mitochondrial genomes have become indispensable tools in species classification, phylogenetic reconstruction, and ecological adaptation studies due to their high mutation rate, maternal inheritance, and lack of recombination. Mitochondrial genes, particularly the cytochrome c oxidase Ⅰ, COⅠ) gene, are widely used in DNA barcoding to facilitate species identification. In evolutionary research, mitochondrial genes are employed to estimate species divergence times, reconstruct phylogenetic trees, and reveal evolutionary histories through molecular clock analyses. Additionally, mitochondrial genomes play a crucial role in studies of adaptation to extreme environments, such as metabolic regulation, gene rearrangement, and selection pressure analysis in extreme ecosystems such as mangroves, coral reefs, and deep-sea ecosystems, thereby helping to elucidate adaptive evolutionary mechanisms. However, mitochondrial genome research in phylogenetics still faces several challenges. Genetic introgression and hybridization can lead to mitochondrial sequence mixing between species, affecting classification accuracy. Incomplete lineage sorting may fail to fully reflect the true evolutionary relationships of species, particularly in rapidly radiating taxa. Furthermore, substitution saturation can result in erroneous phylogenetic topologies, while nuclear mitochondrial pseudogenes (NUMTs) in the nuclear genome can interfere with species identification and phylogenetic inference. Future studies should integrate multi-genomic data, combining nuclear genome, environmental DNA, and metagenomic data to enhance the accuracy of phylogenetic research. Additionally, greater efforts should be made to investigate mitochondrial genomes in understudied ecosystems and to explore adaptive evolution in extreme environments. With advancements in high-throughput sequencing technologies, single-cell genomics, and machine-learning approaches, mitochondrial genome research is expected to play an increasingly vital role in species classification, evolutionary biology, and ecological adaptation studies, providing theoretical support for biodiversity conservation and ecosystem management.