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Non-native species threaten native ecosystems and species, particularly on islands where rates of endemism and vulnerability to threats are high. Understanding species invasion will aid in providing insights into ecological and evolutionary processes. To identify the non-native sika deer (<i>Cervus nippon</i>) population in Jeju, South Korea, and their phylogenetic affinities, we collected tissue samples from roadkill and the World Natural Heritage Headquarters in Jeju. Mitochondrial DNA cytochrome B (<i>CytB</i>) gene sequences were analyzed to determine two distinct <i>CytB</i> haplotypes. Phylogenetic analysis using maximum likelihood <i>tree</i> revealed two haplotypes of <i>CytB</i> clustered into two different groups representing two subspecies: <i>C. n. yakushimae</i>, native to Japan, and <i>C. n. taiouanus</i>, native to Taiwan. The tentative divergence time between the two subspecies was estimated at 1.81 million years. Our study confirmed that the two subspecies of sika deer are sympatric in the natural ecosystem of Jeju Island. This study provides valuable information to help government and conservation agencies understand alien species and determine control policies for conserving native biodiversity in South Korea.