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Jatropha curcas and J. macrocarpa, suitable for production of biodiesel oil from their seeds, are able to live in arid and semi-arid regions, where most crops cannot survive. J. curcas is characterized by higher oil quality and seed yield, but it is not a good candidate for oil production in arid areas with freezing temperatures, due to its sensitivity to chilling in comparison to J. macrocarpa. In this work, for the first time, the effects of cold stress and different mechanisms activated in these conditions have been studied in the two species. Seedlings were treated with low non-freezing temperatures with or without a previous acclimation period. Water status, pigment content, oxidative stress and antioxidant response were studied in acclimated and non-acclimated plants. The key features that differentiate J. macrocarpa from J. curcas were the ability to accumulate, at low temperatures, high concentrations of pigments and glutathione and significantly higher activities of ascorbate peroxidase. These data could explain the greater resistance to low temperatures of J. macrocarpa. A period of acclimation was not able to improve cold tolerance of J. curcas and this confirms its limited adaptability to arid areas with freezing temperatures.