The phylogenetic analysis of 19 taxa of four subgenera of Prunus distributed in Korea (Cerasus, Amygdalus, Padus, and Prunophora), and two outgroup taxa, were analyzed by comparing nucleotide sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA. The lengths of the ITS1 and ITS2 regions varied from 223 to 237 base pairs (bp) and from 230 to 245 bp, respectively. The longest of all the ITS regions was that of P. leveilleana, at 480 bp. The GC content of the selected Prunus species ranged from 58.3 to 64.6% in ITS1 and from 62.0 to 65.3% in ITS2. Pairwise sequence divergence ranged from 0 to 17.5% within the Prunus taxa, and from 24.1 to 53.6% between ingroup and outgroup taxa. Sequence alignment of the ITS region included 529 positions among the 21 taxa. The four subgenera of Prunus were genetically monophyletic. In addition, subgenera Amygdalus and Prunophora were more closely related to each other than to any other subgenus, having a common ancestral divergence point with a bootstrap value of 100%. A remarkable result is that the P. yedoensis wilds from Jeju ended up far from the P. yedoensis cultivars, as was proposed in a previous study based on random amplified polymorphic DNA (RAPD). These results show that ITS sequence analysis is a useful approach for inferring phylogenetic relationships among members of the genus Prunus distributed in Korea and to select wild plants of P. yedoensis.