Water deficit is a major constraint affecting crop productivity, and understanding the responses of different landraces to water scarcity is crucial for sustainable agriculture. This study aimed to investigate the physiological and biochemical responses of watermelon landraces under varying levels of water deficit. Six native watermelon landraces (Aliabad, Sistani, Torbat, Mashhad, Mazandaran, and Gorgan) were evaluated in a split-plot design with three replications, using different irrigation levels (40, 70, and 100% of the available water). The results revealed that drought stress significantly influenced the relative water content (RWC) of watermelon leaves, with the highest decrease observed in severe drought stress (40%) for the Mazandaran and Gorgan landraces. Proline content, an important osmolyte, increased significantly in all landraces under severe drought stress, with the Sistani landrace exhibiting the highest accumulation. Electrolyte leakage increased with increasing drought stress, particularly in landraces from humid regions (Mazandaran and Gorgan). Water use efficiency decreased with higher drought stress levels, but the Sistani landrace maintained higher efficiency due to its consistent yield performance. Antioxidant enzyme activity, including catalase, ascorbate peroxidase, superoxide dismutase, and glutathione reductase, increased in response to drought stress, especially in landraces from dry regions (Sistani and Mashhad). The yield parameters, including fruit weight, size, and yield per hectare, declined with increasing drought stress, but landraces from dry regions demonstrated better performance under severe stress. Under severe drought stress the Sistani landrace exhibited the highest lycopene content, whereas the Mazandaran landrace had the lowest. Total soluble carbohydrates and total soluble solids increased initially and then decreased with increasing drought stress, with the Sistani and Mashhad landraces showing the highest values. This study highlights the potential use of Sistani and Mashhad landraces, due to their superior performance and tolerance to water deficit stress, as genetic resources for cultivation in water-limited conditions and also for watermelon genetic improvement programs.