The raccoon dog (Nyctereutes procyonoides) is distributed from southeastern Siberia to northern Vietnam, including Korea and Japan, as well as Europe. In Korea, most of its predators and competitors are extinct, which has resulted in rapid growth of the raccoon dog population. This population increase has raised concerns about its role in the ecosystem and the zoonotic transfer of various contagious diseases, and thus an effective method of raccoon dog population control in Korea is required. To investigate the genetic diversity and structure of raccoon dog populations, 12 polymorphic microsatellite loci were identified and characterized. These novel microsatellite markers were employed to obtain basic population genetic parameters for 104 N. procyonoides specimens from five locations in South Korea. The mean allele number of 12 loci across samples was 8.7, and the number of alleles per locus ranged 2-13. Mean expected and observed heterozygosities were 0.723 and 0.619, respectively. Genetic differentiation, estimated by pairwise FST, was significant for all population pairs excepting Seoul/Gyeonggi and Gangwon pair, with a moderate level of genetic differentiation for all the population pairs (mean FST = 0.054), but little differentiation between Seoul/Gyeonggi and Gangwon (FST = 0.024). Bayesian-based clustering analysis predicted that Korean raccoon dog population is composed of four distinct genetic subpopulations. These genetic information and structure of raccoon dog will be very useful to prevent spreading infectious diseases.