Gamma-ray-induced mutagenesis and in vitro selection of sugarcane (Saccharum officinarum L. Lam.) plants tolerant to NaCl was achieved using embryogenic calli of sugarcane var. Co740. Sensitivity to gamma radiation and NaCl was studied in vitro in embryogenic calli followed by characterization and evaluation of mutant clones. The results indicated that callus growth and plant regeneration were significantly affected by radiation dose (0, 10, 20, 30, 40, 50, 60, 70, or 80 Gy ⁶⁰Co gamma rays) as well as NaCl exposure (0, 50, 100, 150, 200, or 250 mM). Salt tolerance was achieved by culturing irradiated callus on selection medium with different NaCl concentrations through stepwise in vitro selection. Salt- selected embryogenic callus lines were then analyzed for proline, glycine betaine, Na⁺, and K⁺ contents. Salt-selected plants were grown to maturity, and their agronomic performance was evaluated under normal and saline conditions. Twenty-four mutant clones were characterized for proline, glycine betaine, Na^+, and K⁺ contents. The mutant clones exhibited improved sugar yield with increments in Brix%, number of millable canes, girth, and yield. The results suggest that in vitro culture and induced mutagenesis offer an effective way to enhance the genetic variation in sugarcane.