Drought conditions may have direct or indirect effects on plant physiology, biochemistry, and molecular characteristics. The purpose of this study was to investigate the effects of drought stress on the physiological, biochemical, and molecular responses of three different watermelon cultivars with varying levels of drought tolerance (24: drought resistant, CS: moderately tolerant, and 98: drought sensitive). The cultivars exhibited different responses to cope with water stress according to their tolerance level. Drought induced significant reductions in chlorophyll a, total chlorophyll and carotenoid content and glutation reductase and ascorbate peroxidase activity in the sensitive cultivar unlike in the moderately tolerant and drought resistant cultivars. Additionally, the expression levels of NAC1, NAC2, ORE1, WRKY24, SAG12, SAG13, KCS2, CER1, DREB2A, LTP3, SWEET15, and PYL9 genes were measured using qRT-PCR. The expression ratios of the genes significantly varied depending on the gene location and on the tolerance of the cultivars. Results showed that the physiology and biochemical and molecular pathways of tolerant cultivars change to adapt to drought conditions. Therefore, the drought-resistant cultivar copes with drought stress by increasing proline content and antioxidant enzyme activities, as well as by increasing the expression of specific genes.