Abstract: Reactive oxygen species (ROS), a by-product of photosynthesis and respiration, is increased under many stress conditions, which would result in the peroxidation of the cytomembrane and damage of the photosystem II reaction center. Soluble carbohydrates such as raffinose family oligosaccharide (RFOs) could respond to stress and might be involved in scavenging hydroxyl radicals. Previous work demonstrated that the freezing tolerance and RFO content of populations of Arabidopsis thaliana along the Yangtze River in China were decreased as compared to the Col wild type because of a natural variation of C-repeat binding factor 3 (CBF3). In this study, we examined the transcription profile of 4 populations along the Yangtze River under cold conditions and found a different pathway of scavenging hydroxyl radicals in these 4 populations as compared with the Col ecotype. Also the concentration of ROS was greater in these 4 populations than in the Col ecotype. When the cold-inducible allele of CBF3 from the Col ecotype was transformed into the CQtlx population, from the highest warmest habitat with the lowest freezing tolerance, the freezing tolerance of transgenic plants was improved significantly as compared with non-transgenic CQtlx plants. At the same time, the concentration of RFOs was increased, but that of ROS was decreased. Thus, under cold conditions, CBF3 could induce synthesis of RFOs, probably to scavenge hydroxyl radicals. The higher concentration of ROS and lower freezing tolerance in populations along the Yangtze River as compared with the Col ecotype might be explained at least in part by the natural variation of CBF3 in these populations.