Dybowski's sika deer (Cervus nippon hortulorum) originally inhabited the majority of the Primorsky Krai in Far Eastern Russia, north-eastern China, and Korean Peninsula. At present, only the Russian population seems to be stable, even though this taxon is still classified as endangered by the Russian Federation. Almost 100 years ago, this subspecies, among others, was imported to several European countries including the Czech Republic. We used both mitochondrial (mtDNA; the cytochrome b gene and the control region) and nuclear DNA markers to examine the actual taxonomic status of modern Czech Dybowski's sika population and to compare the genetic diversity between the introduced and the native populations. Altogether, 124 Czech samples and 109 Primorian samples were used in the analyses. Within the samples obtained from individuals that were all morphologically classified as Dybowski's sika, we detected mtDNA haplotypes of Dybowski's sika (84 samples), as well as those belonging to other sika subspecies: northern Japanese sika (25 samples), southern Japanese sika (6 samples), and south-eastern Chinese sika (8 samples). Microsatellite analysis revealed a certain level of heterozygote deficiency and a high level of inbreeding in both populations. The high number of private alleles, factorial correspondence analysis, and Bayesian clustering analysis indicate a high level of divergence between both populations. The large degree of differentiation and the high number of population-specific alleles could be a result of a founder effect, could be a result of a previously suggested bottleneck within the Primorian population, and could also be affected by the crossbreeding of captive individuals with other sika subspecies.