The genetic characterization of Indian sesame cultivars and related wild species was analysed using 102 simple sequence repeat (SSR; microsatellite) markers. Of these, 62 were novel sesame-specific microsatellites isolated in the course of the present investigation by constructing genomic libraries. Characterization of the 68 sesame accessions and three related wild species using 72 polymorphic SSR primers resulted in the detection of 170 alleles. The number of alleles ranged from two to four with an average of 2.5 alleles per locus. Polymorphic information content of the markers ranged from 0.43 to 0.88 with an average of 0.66. UPGMA cluster analysis grouped all the accessions into two major clusters with a genetic similarity ranging from 0.40 to 0.91. A moderate to high level of genetic variability was observed. The three wild accessions used in the study formed separate clades and distant genetic relationships were observed between the cultivar lines and wild species. Differentiation of genotypes according to geographical region was not observed. Analysis of molecular variance (AMOVA) analysis revealed that a high percentage of variation was within populations (87.1 %). An overall Fst of 0.11 among the populations indicated low population differentiation. The SSR markers developed will be useful for further genetic analysis, linkage mapping and selection of parents in future breeding programmes.