An attempt was made to find out the effect of gamma radiation on Panicum sumatrense. The seeds of little millet cv. ATL-1 and CO-2 were exposed to different doses of gamma rays, like 100, 200, 300, 400, 500, and 600 Gy. The seeds treated with gamma rays were sown in plastic pots with red soil to analyze the LD50 value and growth parameters. The lethal dose 50% value was observed at 400 Gy and 500 Gy for CV ATL-1 and CO-2, respectively. In both cultivars, all the growth parameters, such as percentage of germination, percentage of survival, shoot length, root length, fresh weight, and dry weight, showed a decreasing trend towards control with increasing doses of gamma rays, and the reduction was maximum at 600 Gy and minimum at 100 Gy. The cv.ATL-1 seedlings treated with gamma radiation were subjected to estimation of total protein content, and this protein content showed a decreasing trend compared to the control. The number of protein bands and kDa values of cv. ATL-1 were observed by using SDS-PAGE analysis. The kDa value of 132.8 was present only in 400, 500, and 600 Gy. The growth attributes of cultivar ATL-1 were unique compared to cultivar CO-2.