Chilli (Capsicum annuum L), a member of Solanaceae is a significant vegetable cum spice crop with immense economic importance cultivated in tropical and sub-tropical regions of the world. Treated the chili seeds with 0.1% EMS for 6, 12, and 24 hrs. The seeds were germinated on fertile soil for two generations. They studied various parameters such as seed germination, plant height, number of fruits per plant, fruit length, number of branches, number of leaves, etc,. Plants of M3 generation were used for molecular study. During molecular studies, DNA is isolated from M3 generation selected mutant seedlings and amplified with ISSR primers. Out of Seven selected primers, three primers such as ISSR-2, ISSR-3, and ISSR-9 showed significant polymorphism in the plants treated for 6 and 12 hrs. A total of 134 bands scored of which 35 bands were polymorphic with an average of 26.11 percent polymorphism. Out of three ISSR-3 showed the highest polymorphism (56.00%) and the primer ISSR-9 gave the lowest polymorphism (17.54%). Significant variations in the banding pattern were noticed in the seven mutant lines compared to the control plant.