The cultivated apple (Malus domestica) is one of the most important fruit crops cultivated in different regions of the world including Iran. For production and breeding of high quality apple, the knowledge of genetic diversity of the cultivated apples is necessary. Therefore we studied genetic diversity of 25 genotypes by using ISSR molecular markers. In present study, ten ISSR primers produced 85% polymorphism. (AGC)5GG and (GA)9C primers produced the highest number of bands, while primers (GA)9A, (GA)9A and UBC 807 produced the lowest number. Some of the genotypes showed presence of specific bands. PCoA (principal co-ordinate analysis) plot of genotypes based on geographical origin (Netherland, France, Lebonan and Iran) grouped cultivars from different geographical regions together and intermixed. UPGMA (unweighted paired group method with arithmetic average) and NJ (Neighbor Joining) dendrograms produced 4 major clusters with few subclusters in each of them indicating intra-group genetic diversity. AMOVA (Analysis of molecular variance) test performed on molecular features of the genotypes present in the 4 clusters revealed significant difference among the clusters. This high value of within clusters genetic variation is in agreement with subclusters formed in each major cluster due to intra-population group variation. STRUCTURE plots of k = 2-7 were obtained and the best groupings was done by k =7. It indicated differences in allelic composition and frequency of the subclusters. The results showed presence of high level of genetic diversity in apple samples studied which can be used in planning further selection and hybridization of apple trees.