Amphitetranychus viennensis (Zacher) is a serious economic pest of various fruit trees. Due to its restricted geographic distribution in Asia and Europe, it is an important quarantine species in the North American countries. Despite its significance in agricultural damage and quarantine, the genetic diversity of this species has not been studied in detail. In this study, we determined the nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA in A. viennensis collected from various regions of Korea. Variations in the COI and ITS2 sequences of the Korean samples ranged up to 6.92 and 4.02 %, respectively. A sequence comparison showed that the Korean samples were divided into two groups, one comprising the Geoje and Jinju samples, and the other comprising all samples except for the Geoje/Jinju samples. In most samples, except for the Geoje/Jinju samples, variations in both COI and ITS2 sequences were less than 1.04 and 0.99 %, respectively. However, the variations between two groups were 6.23–6.92 % and 3.02–4.02 %, respectively. In addition, the phylogenic analysis performed with sequences in the GenBank database showed that the samples from Geoje/Jinju, which are located in the southern region of Korea, were most similar to the Japanese samples, but the others were most similar to those of China and Taiwan. Thus, our study suggests that two genetically different groups of A. viennensis exist in Korea.