Native sika deer (Cervus nippon) had not been observed in and around Sasebo City, Nagasaki Prefecture, Kyushu, Japan. However, deer have recently been confirmed in this area, and the number of individuals and the range have been expanding, causing damage to forestry and natural vegetation. Although the origin of these deer is unknown, it may be derived from Formosan sika deer (C. n. taiouanus), which is listed as an invasive alien species in Japan. There is concern about hybridization between this subspecies and the native Japanese sika deer. In the present study, we used genetic methods to assess the current status of the deer in the Sasebo area of Nagasaki Prefecture, particularly its origin and hybridization. Our study showed that the deer did not originate from Formosan sika deer, and we found no evidence of hybridization. The deer in the Sasebo area were found to have a haplotype included in the southern Japanese sika deer lineage. The current study was unable to identify the specific origin of the deer due to limitations in PCR-RFLP and mitochondrial DNA sequencing analysis.