The Japanese oak silkmoth, Antheraea yamamai Guérin-Méneville, 1861 (Lepidoptera: Saturniidae), is an important natural resource of industrial value for silk fiber production. Owing to a lack of geographic and population genetic information, systematic domestication of An. yamamai has not been possible yet. In this study, 10 microsatellite markers developed using next-generation sequencing and two mitochondrial DNA (mtDNA) gene sequences (COI and ND4) were used to investigate the genetic variation and geographic structure of An. yamamai populations in South Korea. The two mtDNA gene sequences revealed very low total genetic variation and, consequently, low geographic variation, validating the use of more variable molecular markers. Genotyping of 76 An. yamamai individuals from nine localities in South Korea showed that the observed number of alleles at each locus ranged from 3 to 26, the polymorphism information content was 0.2990-0.9014, the observed and expected heterozygosities were 0.3252-0.9076 and 0.2500-0.9054, respectively, and F was -0.654-0.520. The population-based F, F, R, and global Mantel tests all suggested that the An. yamamai populations were overall well-interconnected, suggesting that any population can be used as a genetic source for domestication. Nevertheless, STRUCTURE analyses using microsatellite data and mtDNA sequences indicated the presence of two genetic pools in many populations, although a plausible explanation for this observation requires further studies.