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The Sambar is one of the largest deer species distributed mainly in Asia, and it has been listed as a vulnerable species. Taxonomy based on morphological characterization has been the gold standard method used to identify the Sambar deer species. Yet, morphological identification is challenging and requires expertise. To conduct species identification and taxonomic decisions, we performed the molecular identification of <i>R. unicolor</i> found in Sri Lanka using DNA barcodes, <i>COI</i>, and <i>Cyt b</i> to compare the Sri Lankan <i>R. unicolor</i> with the Indian <i>R. unicolor</i> and other <i>R. unicolor</i> subspecies. We obtained mitochondrial DNA sequences from <i>COI</i> and <i>Cyt b</i> from blood samples collected from the wet zone in Sri Lanka. A phylogenetic tree was constructed based on the Bayesian analyses using MrBayes 3.2.7. Molecular dating was implemented in Bayesian Evolutionary Analysis Sampling Trees (BEAST v1.8.2) on the concatenated sequence using a log-normal relaxed clock and Yule species tree prior, with four categories. The results showed that the Sri Lankan <i>R. unicolor</i> is genetically different from the Indian <i>R. unicolor</i> and other <i>R. unicolor</i> subspecies. The divergence occurred approximately 1.1 MYA (million years ago) in the Pleistocene era. The results are essential for designing new conservation platforms for these Sambar deer species.