Sesame is one of the oldest cultivated plants by human being and due to its high quality aromatic seed oil has been named the Queen of the oily seeds. Information on the degree of genetic variation can help improve this valuable crop. One of the main uses of molecular markers in plant breeding is their application in assessment of plant genetic diversity. In the last decades different molecular markers have been developed and used extensively in plant breeding programs. The main uses of the molecular markers are in the finger printing of varieties, allele introgression and study of genetic variation in many plants. In this research genetic variation of 46 sesame genotypes collected from different areas of Iran was assed using both Simple Sequence Repeat (SSRs) and Inter Simple Sequence Repeats (ISSR) primers. Inter-simple sequence repeats markers are widely used in genetic diversity studies because they need no prior DNA sequence information and their development costs are low. From 20 used ISSR primers, 16 produced polymorphic bands. In total 140 scorable bands with the size range of 200 to 2000 base pair were produced. Two primers with 14 polymorphic bands produced the highest and one primer with 3 polymorphic bands produced the lowest number of scorable bands. Primers 222 and b6 respectively had the most and the least polymorphism information content (PIC). Gene variety was 0.27 and Shanon information index was calculated to be 0.42. The mean of fragments for each primer in the studied genotypes was 7/87. Using NTSYS pc 2.02 with Jaccard similarity matrix and UPGMA method a dendrogram was drawn according to ISSR data of genotypes. Base on this analysis the genotypes were categorized in to 5 main groups. In addition to ISSR primers 10 SSR primers were also used to study the genetic variation of the 46 sesame genotypes. Of the 10 pair of SSR primers used only 8 pairs created polymorphic PCR bands. In total 30 bands (allele) were created and scored. The number of alleles propagated by each of these primer pairs varied from 2 to 6 with an average of 4 alleles per locus. The size of amplified fragments ranged from 150 to 300bp. The rate of heterozygosity, polymorphism information content (PIC) and the gen diversity in the studied genotypes were 66%, 51% and 58% respectively. Using NTSYS pc 2.02 with Jaccard similarity matrix and UPGMA method a dendrogram was drawn according to SSR data of genotypes. Base on this analysis the genotypes were categorized in to 9 main groups. Cluster analysis and Principal component analysis for two molecular marker systems showed that most genotypes were not grouped based on their geographical distribution. Key words : sesame, genetic diversity, microsatellite