Rusa unicolor has been listed as a vulnerable species by the International Union for Conservation of Nature and Natural Resources because of human activities. In recent years, population numbers have decreased due to heavy hunting and habitat loss, and little genetic data on this species exists; thus, our knowledge of range distribution and population size remains limited. In the current study, the complete R. u. cambojensis mitochondrial genome was sequenced using polymerase chain reaction followed by direct sequencing. The complete mitochondrial genome was determined to be circular and contain 16,557 bp, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 control region, the gene composition and order were similar to those of most other vertebrates reported to date. Most mitochondrial genes, except for ND6 and eight tRNAs, were encoded on the heavy strand. The overall base composition of the heavy strand was 33.6% A, 28.9% T, 24.2% C, and 13.3% G, with a strong AT bias of 62.5%. There were 13 regions of gene overlap totaling 96 bp and 12 intergenic spacer regions totaling 70 bp. The phylogenetic analyses (maximum likelihood and Bayesian inference) of R. unicolor based on the mitochondrial genome four subspecies of R. unicolor were clustered into a well-supported single clade, and R. u. cambojensis was most closely related to R. u. dejeani. This study will assist in the exploration of the evolutionary history and taxonomic status of the sambar, as well as its protection as a genetic resource.