Tomato is one of the popular vegetables in the world. However, drought has become more serious in recent years. Drought strongly reduced the development and yield of tomato. Thus, the effect of drought stress condition (½ Murashige and Skoog’s medium supplemented with 35 g.L-1 mannitol) and thermal pre-treatment (45 (C for 120 min) on in vitro shoot development of Solanum lycopersicum L. were studied to improve the drought-tolerant of tomato. Morphological, physiological and biochemical changes during the development of in vitro shoot in the drought stress condition were analyzed. Then, leaves of the shoot that pre-treated at 45 (C for 120 min and developed on the drought stress condition were cultured on ½ MS medium supplemented with 20 g.L-1 sucrose, 0.1 mg.L-1 Indole acetic acid, 2 mg.L-1 zeatin and 35 g.L-1 mannitol to regenerate the drought-tolerant shoots. In the drought stress condition (½ MS medium with 35 g.L-1 mannitol), the height of shoot, the number of leaves, and the total leaf area were decreased by approximately 50 % compared to control (½ MS medium without mannitol). Besides, the leaf midrib thickness and the vascular bundle width of the leaf in drought stress were smaller than those of the control. The leaf midrib thickness and the leaf vascular bundle width in drought stress condition were 953.30 (m and 320.21 (m, compared to 1243.80 (m and 410.15 (m in that of control. Chlorophyll fluorescence occurs in some parenchyma cells near the midrib vascular bundle of treated leaves instead of all parenchyma cells in control leaves. Under the drought stress condition, chlorophyll content, photosynthetic intensity and cytokinin activity of leaf were strongly decreased but respiration intensity, the content of carotenoid and proline, especially the activity auxin and abscisic acid were increased. Thermal pre-treatment stimulated shoot regeneration (2.53 shoots/leaf explant) while there was no shoot regenerated from the leaf of the control plant. The shoots regenerated by this method grew well in the drought stress condition.