Inter-simple sequence repeat (ISSR) markers were used to study the genetic diversity and phylogenetic relationships among 16 genotypes from subgenus Prunus (six genotypes from section Prunophora, seven genotypes from section Armeniaca and two plumcot genotypes, and one genotype from subgenus Cerasus) in Prunus genus. From the polymerase chain reaction amplifications with 20 ISSR primers showing polymorphism among subgenera and sections, 180 polymorphic ISSR bands were detected and polymorphism ratio ranged from 57% to 100%. Based on the unweighted pair group method with arithmetic mean (UPGMA) analysis and principal coordinate analysis (PCoA) using the Jaccard coefficient, a dendrogram and three-dimensional plot were constructed including genotypes in Prunus genus. Two main groups formed in the dendrogram; one of them (Cluster I) included Cerasus, whereas Cluster II included Prunus. Cluster II also divided into three subgroups, including sections Prunophora, Armeniaca, and plumcot. Both UPGMA and the PCoA demonstrated that Armeniaca genotypes had lower genetic variation and plumcot genotypes are closer to the plums than the apricots. The ISSR-based phylogeny was generally consistent with Prunus taxonomy based on molecular evidence, suggesting the applicability of ISSR analysis for genotypic and phylogenetic studies in Prunus genus.