Resumo. This work was carried out to evaluate the effects of the rustification on cold tolerance in Eucalyptus dunnii Maiden, Eucalyptus benthamii Maiden & Cambage, Eucalyptus grandis W. Hill ex Maiden, and Eucalyptus saligna Sm., by quantifying leaf contents of total soluble carbohydrates and proline. Seedlings (three months old and about 50 cm height) of these species were submitted to two periods of rustification (zero and 21 days exposure to day/night temperatures of 5 ºC/1 ºC), with photoperiod of 12 hours. After each period of rustification, the seedlings were submitted, for 3 hours, to three temperatures below 0 ºC (-2 ºC, -5 ºC, and -8 ºC). The seedlings were then assessed for leaf contents of total soluble carbohydrates and proline, lethal temperature of 50 % (LT50), and cold damage index. Proline was not detected in the leaves of all species. However, in non-rustified seedlings, there were differences between the species regarding to the foliar contents of carbohydrates, with the highest values in Eucalyptus benthamii, intermediary in Eucalyptus dunnii and Eucalyptus grandis, and the lowest in Eucalyptus saligna. The rustification treatment increased leaf concentration of total soluble carbohydrates by 2.9, 2.5, 2.8, and 1.3 in Eucalyptus dunnii, Eucalyptus benthamii, Eucalyptus saligna and Eucalyptus grandis, respectively. In the seedlings not submitted to the rustification, the LT50 value was lower in Eucalyptus benthamii, intermediary in Eucalyptus dunnii and Eucalyptus grandis, and higher in Eucalyptus saligna. However, in rustified seedlings the LT50 was no different among the species. Rustification reduced the LT50, except in Eucalyptus benthamii. There was a negative correlation between leaf concentration of total soluble carbohydrates and the LT50, considering all Eucalyptus species evaluated. The results show that leaf concentration of total soluble carbohydrates can be used as an indicator of cold tolerance in species of Eucalyptus.