One of the key sources of genetic variability that is ultimately used in the plant development program is mutations. In order to improve crops, mutagens are used, and mutants have been obtained from induced mutations. Gamma irradiation is one of the mutagens that helps to quantify the frequency and patterns of alterations in particular plants. In this present 26 study, the gamma irradiation effect was investigated by germination study and physiological characteristics of green gram. Seeds were exposed to a gamma source (CO60) at doses ranging from 100, 200, 300, 400, 500, 600, 700, and 800, with a non-irradiated sample as a control. Our results show that the germination percentages as well as morphological parameters of seedlings (root length, shoot length, fresh weight, and dry weight) decreased with increasing irradiation doses. These findings were confirmed by ESR spectroscopy with the g-factor at 2.000 ± 0.005. The photosynthetic pigments such as chlorophyll a and b were decreased in irradiated seedlings compared to control, where carotenoid content was increased. Biochemical content such as protein, amino acid, reducing sugar, starch and proline content was increased with increasing doses and it was concerned with various stretching bands analyzed by FTIR analysis. MDA content was increased with increasing doses by the production of free radicals. Antioxidant enzymes such as catalase and peroxidase were increased in irradiated seedlings, which act as free radical scavengers. These results suggested that there had been up to 600 Gy significant changes in morphology, photosynthetic pigment, biochemistry, and antioxidant analyses.