It is well demonstrated that wheat-rye 1BL/1RS translocated chromosome leads to some valuable novel traits such as disease resistance, high yield and functional stay-green after anthesis. To understand the physiological mechanism of 1BL/1RS translocation responsible for osmotic stress, two wheat cultivars, CN12 and CN17, carrying the translocated chromosome and MY11 without the translocated chromosome were employed in the study. During 5-day osmotic stress, fresh weight inhibition, chlorophyll content, soluble protein content, MDA concentration, antioxidant enzymes activity and free polyamines content were examined. CN12 and CN17, especially cultivar CN17, registered greater biomass and minor oxidative damage compared with their wheat parent. Meanwhile, the concentration of Spd and Spm in CN17 was significantly higher than the others. In addition, we found a positive correlation of fresh weight inhibition (FWI) and Put concentration, and a negative one with the parameters (Spd + Spm): Put ratio, indicating the importance of higher polyamine (Spd and Spm) accumulation on the adaptation to osmotic stress. Therefore, we proposed that the accumulation of higher polyamines (Spd and Spm) should play an important role on the adaptation of 1BL/1RS translocation lines to osmotic stress and might be important factors for the origin of novel traits introduced by 1BL/1RS.