Species identification is one of the critical challenges, which researchers in the animal investigation are facing under various objective scopes, such as cell culture in medicine, the ancient remainder in archaeology, traditional medicine conservation, ecology, biodiversity studies, wildlife criminal science, and food industry. Recently, various strategies have been developed, but specifically, genome-based methods for the identification of species are preferred over protein-based experiments. The reasons include but are not limited to sample damaging and deficiency or closely related species. Although novel approaches, such as next-generation sequencing (NGS), barcoding, loop-mediated isothermal amplification (LAMP), and droplet digital PCR (ddPCR) have been developed, still traditional methods of RFLP, Nested PCR, Multiplex PCR, and Real-time PCR are in wide use. Most of the above-mentioned methods have utilized conserved mitochondrial genomic regions such as cytochrome c oxidase subunit I (COI), Ribosomal DNA (rDNA), D-Loop, NADH, and cytochrome b for species identification. It was accepted that species identification from a small amount of the target sample is still a challenge. Hence, this presentation proposes a diverse application of high mitochondrion gene copy number per cell as an effective way for species identification with a small amount of targeted gene. Furthermore, a relatively high mutation rate can be found in this method in comparison to nuclear DNA, which helps to determine closely related animal species. This review discusses a broad range of species identification applications by the mitochondrial gene-based strategies, which can provide an exciting perspective for future research in animal investigation.