Mutation induction with chemical mutagen ethyl methane sulfonate (EMS) capable of producing genetic variation in plants. Various concentration of EMS (0%; 0.01%; 0.02%; 0.04%) were applied to Indonesian local chili pepper Genotypes 2, 7, and 11. Genetic variation among three genotype of chili pepper was assessed using three SSR primers namely CA26, CA52 and CA96. A total of 18 alleles were identified for the three SSR loci with an average Polymorphic Information Content (PIC) value of 0.829. Three genotype of chili pepper had different sensitivity to EMS mutation induction. Genotype 11 plants showed higher sensitivity to EMS treatment than Genotypes 2 and 7. Dendrogram constructed based on Jaccard's similarity coefficient was divided chili pepper mutants and control plants into three main clusters. Similar genotype either control or mutants plant, especially Genotypes 2 and 7 were grouped into similar cluster. However, large genome changes in Genotype 11 caused mutant plants G11K1, G11K2, G11K3 had low genetic similarity to their control plant, so the mutants were separated in a different group from the control plant. This study revealed that EMS mutation induction capable of increasing genetic variation in chili pepper plants based on SSR molecular marker analysis.