A well-informed choice of genetic locus is central to DNA barcoding.Current DNA barcoding in animals involves the use of the 5’ half of themitochondrial cytochrome oxidase 1 gene (CO1) to diagnose and delimit species.However, there is no compelling a priori reason for the exclusive focus on this region,and it has been shown that it performs poorly for certain animal groups. To explore alternative mitochondrial barcoding regions, this research compared the efficacy of the universal CO1 barcode with the other mitochondrial protein-coding genes based on 1,179 mitochondrial genomes of eutherians via bioinformatic analyses. Results from analyses on the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation show that the universal CO1 barcode is a good representative of mitochondrial genes, because the high species-recovery rate (>90%) was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. However, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. And any choice of mitochondrial gene for DNA barcoding failed to offer significant resolution at higher taxonomic levels. On the whole, the research suggests that the universal CO1 barcode is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification.