Sesame is an important oil seed crop containing high amount of edible oil along with nutritional quality. A wide range of colours is observed in the sesame seeds ranging from white to black. White seeded sesame has huge demand in confectionary market besides the oil market. However, yield level of sesame is low compared to other oilseed crops. Improvement of sesame in terms of yield and quality is only possible by utilization of genetic variations. The present investigation was conducted at Experimental Farm, Agricultural College and Research Institute, Madurai during 2018 and 2019 to create novel genetic variations using gamma ray and combination of gamma ray + EMS as mutagens. The white seeded sesame variety VRI 3 was treated with gamma ray viz., 300 Gy, 350 Gy, 400 Gy and gamma ray + EMS combination treatments viz., 300 Gy + 10 mM, 350 Gy + 10 mM and 400 Gy + 10 mM. In the first generation, the mean performance for morphological and physiological traits decreased with increase in dose of mutagens; while biochemical traits increased at lower doses and reduced at higher doses. High effectiveness for chlorophyll mutants was recorded in 400 Gy gamma ray treatment. Gamma ray treatments were found to induce higher number of macro mutants than combination treatments. From this study it is concluded that biochemical traits get enhanced in lower doses of gamma ray treatment and gamma ray treatment is more suitable for induction of viable mutants than combination treatments.