The objective of this research was to evaluate the genetic diversity among twelve apple rootstocks by using 14 simple sequence repeat primer pairs. Generally, 44 alleles were scored at SSR loci. The number of alleles (na) per locus ranged from two to five alleles with an average of 3.14. The maximum effective number of alleles (ne), expected heterozygosity (He), and observed heterozygosity (Hobs) were 2.3, 0.56 and 0.36, respectively. Results showed that the studied genotypes were not in Hardy-Weinberg equilibrium in most of studied loci. The polymorphism information content (PIC) varied from 0.5 to 0.86 which confirmed the effectiveness of SSR markers in determination of molecular polymorphism and characterization of the rootstocks genotypes. Cluster analysis using UPGMA algorithm on the basis of Jaccard’s similarity coefficient classified the studied genotypes into three distinct groups. In this study, three Iranian local rootstocks were located in separate groups.