Jerusalem artichoke could be an alternative feedstock for bioenergy during times when there are shortages of other raw materials for the ethanol industry. However, insufficient water under rainfed conditions is a major cause of Jerusalem artichoke losses. Genetic variation for drought tolerance is an essential prerequisite for the development of Jerusalem artichoke cultivars with improved drought tolerance. The objectives of this study were to determine the effects of drought stress on tuber dry weight and biomass and to investigate the genotypic variability in Jerusalem artichoke germplasm. The line-source sprinkler technique was used to compare moisture responses of a range of 40 Jerusalem artichoke genotypes grown using 3 water levels. Experiments were conducted on a Yasothon soil series in Northeast Thailand during 2010/11 and 2011/12 and included extended dry periods. Drought reduced tuber dry weight and biomass, and the reductions in tuber dry weight and biomass were greater under severe drought than moderate drought conditions. Over both seasons, CN 52867, HEL 53, HEL 231, HEL 335, JA 76, HEL 65, and JA 102 × JA 89 (8) had consistently high tuber dry weight (1.3 to 4.5 t ha-1) and HEL 53, HEL 61, HEL 231, HEL 335, JA 76, JA 15, JA 89, HEL 65, HEL 256, and JA 102 × JA 89 (8) had consistently high biomass (2.0 to 6.8 t ha-1). These Jerusalem artichoke genotypes are promising parents in breeding for drought tolerance.