In vitro selection of drought-tolerant cassava varieties is essential for rapid breeding for drought tolerance. The objectives of this study were to determine the response of three contrasting cassava varieties to mannitol-induced drought stress to establish its suitability for in vitro screening and examine relationships among growth parameters. Plantlets were raised from nodal segments on Murashige and Skoog (MS) medium containing 0 (control), 5, 10, 15, 20, 25 and 30 g/l mannitol. Variety CH 140 had the highest survival of explants and frequency of root formation, while MV 99/0395 recorded the highest number of chlorotic leaves and the lowest survival of explants. The lowest numbers of leaves were produced at 25 and 30 g/l mannitol by the three varieties. In CH 140, the highest number of leaves was produced in medium free of mannitol, while the highest number of leaves was produced at 5 and 10 g/l mannitol in MV 99/0395 and TMS 01/1206, respectively. In TMS 01/1206, number of roots produced decreased as the concentration of mannitol in culture media increased, whereas in CH140, number of roots increased as the concentration of mannitol increased before decreasing; while in MV 99/0395, number of roots was not affected by an increase in mannitol concentration. As the concentration of mannitol in the culture media increased shoot height of plantlets decreased with a sharp decline at 20 mg/l mannitol. Concentration of mannitol and survival of explants had significant negative correlation with all parameters. However, frequency of root formation only had significant positive correlation with shoot length. The study concluded that differential responses were expressed by the three varieties to mannitol-induced drought stress and mannitol at 20 g/l concentration was a suitable in vitro drought inducing-agent for screening cassava varieties for drought tolerance.