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Drought has been recognized as a potential challenge to maize production around the world, particularly in arid and semi-arid regions. The primary focus of the present study was to investigate the metabolic and physiological adjustment mechanisms as well as drought-responsive gene expression patterns in five maize (Zea mays L.) genotypes (G314, G2, G10, G123, and G326) with varying drought-tolerance capacities at the vegetative stage. Twenty-one days-old maize plants from five maize genotypes were submitted to a well-watered (10 days) watering interval as a control, mild water stress (15 day interval), and severe water stress (20 day interval) treatments in a field experiment for two successive seasons (2019 and 2020). For all maize genotypes, the results showed that water stress significantly reduced plant height, leaf area, biomass, and yield characteristics. However, water stress, which was associated with the length of the watering interval, increased the concentrations of glycine betaine, amino acids, proline, phenols, flavonoids, soluble proteins, and soluble sugars, as well as catalase and peroxidase activities. On the transcriptional level, prolonged water stress increased the expression of drought-responsive genes (LOS5, Rad17, NCED1, CAT1, and ZmP5CS1), with G10 and G123 genotypes being the most drought-resistant. Herein, genotypes G10 and G123 were shown in this study to be relatively water stress tolerant due to improved osmoregulatory, antioxidant, and metabolic activities under water stress conditions, as well as the fact that they were endowed with stress-responsive genes.