Sesamum indicum L. (family: Pedaliaceae) is an economically important oil seed crop grown in tropical and sub-tropical countries. It is widely used in food, nutraceutical, pharmaceutical industries. Sesamum is widely distributed in all the climatic stages and great diversity. The exploration of genetic diversity is a pre-requisite for genome organization in the landraces and the related domesticated ones. Agromorphological and molecular markers were used to assess the identification of 33 Sesamum genotypes and determination of the genetic relationships among these genotypes. Out of 30 Inter-Simple Sequence Repeat (ISSR) primers tested, 18 primers produced 114 detectable fragments, of which 97 (85.08%) were polymorphic across the varieties. Molecular profiling could be solely used for their identification of genotypes. Genetic relationships among these genotypes were evaluated by generating a similarity matrix based on the Jacard's coefficient and the Unweighted Pair Group Method with Arithmetic Average (UPGMA) dendogram. The results showed a clear cut separation of the 33 genotypes and were in broad agreement with the morphology. Both molecular and morphological markers will be useful for preservation of the germplasm as well as breeding program.