Prunus subgenus Cerasus is a large and diverse genus. Its botanical classification has long been controversial and complicated. Molecular markers derived from the chloroplast genome may provide useful tools for phylogenetic resolution and taxonomic study in Prunus species. Thus, we compared chloroplast genomes of 23 Prunus species to identify sequences with high variation levels. The repeated sequences (RSs) and two types of sequence variation, comprising divergent homologous regions and short sequence repeats (SSRs), were identified. The species with the highest number of RSs was P. emarginata (60), whereas P. campanulata contained the highest amount of SSRs (70). In contrast, P. dictyoneura and P. yedoensis contained the lowest number of RSs (37) and SSRs (46), respectively. Out of these homologous SSRs, 11 were shared in the 23 species, and seven of them showed variations. A total of 203 homologous regions were identified. Out of these, 20 regions (19 IGSs and one intron) showed a high variation from 6.75 to 19.31%. The phylogenetic tree which was inferred based on 19 highly variable regions showed a topology similar to the one obtained using the whole chloroplast genome. We conclude that the seven variable SSRs and the 20 highly variable regions may potentially be convenient as molecular markers for phylogenetic and population genetics studies in subgenus Cerasus species. In addition, the molecular phylogenetic position of P. clarofolia was inferred based on the 19 highly variable regions.