Asian Journal of Research in Zoology

Mitochondrial DNA (mtDNA) remains a widely used molecular resource in animal systematics, phylogeography, population genetics, conservation biology, and biodiversity assessment. Its high cellular copy number, compact genome, and informative variation across loci such as cytochrome c oxidase subunit I, NADH dehydrogenase genes, 12S and 16S rRNA, and the control region support applications ranging from species identification to population-level inference. This narrative review synthesises the principal applications of mtDNA in phylogenetic reconstruction, phylogeography, population genetics, DNA barcoding, aquatic biodiversity monitoring, environmental DNA metabarcoding, conservation, and mitogenomics. It also examines major biological and analytical limitations, including maternal inheritance, heteroplasmy, nuclear mitochondrial DNA segments, introgression, mitochondrial capture, incomplete lineage sorting, natural selection, inherited symbionts, and mito-nuclear discordance. Particular attention is given to the influence of reference-library quality, marker choice, sampling design, and analytical quality control on interpretation. The review further considers bioinformatic workflows, historical DNA, next-generation sequencing, population mitogenomics, and emerging machine-learning applications, while emphasizing that analytical tools cannot compensate for weak sampling, contamination, or incomplete reference data. Complete mitochondrial genomes can improve character sampling and phylogenetic resolution, but they remain linked and therefore retain the inheritance constraints of a single mitochondrial lineage. Accordingly, mtDNA is most informative when its results are interpreted with nuclear genomic, morphological, ecological, and geographic evidence. An integrated approach can reduce overinterpretation and support more robust assessments of evolution, taxonomy, conservation, and biodiversity.