The genetic diversity and phylogenetic relationships of sika deer of different subspecies are uncertain. In order to explore the phylogenetic relationship of different sika deer subspecies, this study used a wider sample collection to analyze mitochondrial sequences and nuclear microsatellites of sika deer. The full lengths of cytochrome-b gene of 134 sika deer were sequenced, and 16 haplotypes were obtained. Based on phylogenetic and haplotype networks analysis, the sika deer was not clustered according to subspecies but was divided into four lineages. Lineage I includes individuals from C.n.kopschi, C.n.sichuanicus, and C.n.hortulorum subspecies; Lineage II includes individuals from C.n.hortulorum subspecies; Lineage III includes individuals from C.n.centralis, C.n.yakushime, C.n.mageshimae, and C.n.keramae subspecies, namely southern Japanese population; Lineage IV includes individuals from C.n.centralis and C.n.yesoensis subspecies, namely northern Japanese population. The microsatellite analysis showed that the sika deer in China and Japan originated independently. The three subspecies of China have significant genetic differentiation, while the three subspecies of Japan have no significant differentiation. This study provides reference for the research of genetic diversity and phylogenetic relationship of sika deer, and also provides scientific data for the evaluation, protection, and utilization of sika deer resources.