Abstract: Background and Objective: Mutagenesis is the process in which genetic information in an organism is changed, not by genetic segregation but induced by chemical or physical agents. The experiment was conducted to investigate the effects of gamma irradiation as physical mutagens on E. grandiflorum plant. Materials and Methods: The experimental study was carried on Eustoma grandiflorum plant to evaluate the effects of gamma radiation as physical mutagenesis at different doses (0, 2, 5, 10, 20, 40, 60, 80, 100 and 120 KR) using Co60 gamma source for 30 min on various morphological, flowering and anatomical characters by Duncan’s New Multiple Range test at 0.05% level of probability. Results: In vitro shooting and rooting behaviors showed the best results with low gamma irradiation (10 KR), while the longest shoots and the highest number of leaves were obtained with gamma dose 60 KR. The highest number of roots and the longest roots were observed with 20 KR of gamma radiation. Highest survival percent of acclimatized plants (63%) was obtained with 10 and 20 KR treatments. A wide range of leafs and flowers colour and form changes were observed after radiation with different doses of gamma rays. Different leaf anatomically structure features that depend on the level of gamma radiation treatments were recorded. Total proteins were extracted from leaves after flowering and analyzed by SDS-PAGE and revealed 26 protein bands with 100 KR and absence with other doses or control. Conclusion: It was concluded that, Gamma radiation with different doses had a potential effect on the production of mutant E. grandiflorum plants due to changing in flowers colour and form.