Abstract: Cut cotyledons of Stylosanthes guianensis cv. Reyan 2 seedling plants were used for explant production based on in vitro callus multiplication techniques. DES mutagenesis was applied under different temperatures in combination with in vitro selection on L-Hyp containing medium for induced callus production. The survivors from this 2 step in vitro selection of L-Hyp to plantlets, were subjected to 1℃-8 d low temperature selection, and the plantlets were then classified according to chilling injury into 4 grades. Randomly selected two plantlets with the lowest level of chilling injury (1^st chilling injury), were taken at random and part of the stems were cut off for callus induction. These calluses were tested for physiological indices of cold resistance. The survival rate of calluses treated at the lowest temperatures was higher under the same concentrations of DES. Treatment with 0.4% DES (10℃, 2 h) for callus had the best survival rate (18.6%),which was significantly better than the 6.6% of the CK. Fourteen plantlets of the 1^st chilling injury were obtained from those that survived the 2 step in vitro selection of L-Hyp after low temperature selection, while there were no surviving plantlets from the CK. The 1^st chilling injury plantlets had a significantly better cold tolerance than those from the CK.