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The mitochondrial genome is able to provide important information for evolutionary biology. Therefore, this research aims at characterizing that of Bubalus quarlesi mitochondrial genome. Furthermore, it was entirely 16,354 bp in length, consisting of 13 protein coding genes (PCGs), 22 tRNA, and two rRNA genes, one putative control region. All PCGs, however, used a typical ATN as start codon, while the stop codon were typically TAA, TGA, and TGA, with the exception of nad3 and nad6 genes using TCA. It was also observed that the lowest evolution rate of PCG was cox1, while the highest was atp8. Meanwhile, a phylogenetic analysis based on 13 PCGs was performed on 14 bovine species, using Bayesian and maximum likelihood, and its tree indicated that B. quarlesi belongs to the subtribe of Bubalina, closely related to Bubalus depressicornis. These, therefore, provide novel meaningful insights for the conservation genetics and population genetics of B. quarlesi.