Analyzing differences in tolerance to drought in Quercus spp., and the characterization of these responses at the species and individual population level, are imperative for the selection of resilient elite genotypes in reforestation programs. The main objective of this work was to evaluate differences in the response and tolerance to water shortage under in five Quercus spp. and five Andalusian Q. ilex populations at the inter- and intraspecies level. Six-month-old seedlings grown in perlite were subjected to drought treatments by withholding water for 28 days under mean 37 °C temperature, 28 W m−2 solar irradiance, and 41% humidity. The use of perlite as the substrate enabled the establishment of severe drought stress with reduction in water availability from 73% (field capacity) to 28% (dryness), corresponding to matric potentials of 0 and −30 kPa. Damage symptoms, mortality rate, leaf water content, photosynthetic, and biochemical parameters (amino acids, sugars, phenolics, and pigments) were determined. At the phenotypic level, based on damage symptoms and mortality, Q. ilex behaved as the most drought tolerant species. Drought caused a significant decrease in leaf fluorescence, photosynthesis rate, and stomatal conductance in all Quercus spp. analyzed, being less pronounced in Q. ilex. There were not differences between irrigated and non-irrigated Q. ilex seedlings in the content of sugar and photosynthetic pigments, while the total amino acid and phenolic content significantly increased under drought conditions. As a response to drought, living Q. ilex seedlings adjust stomata opening and gas exchange, and keep hydrated, photosynthetically active, and metabolically competent. At the population level, based on damage symptoms, mortality, and physiological parameters, the eastern Andalusian populations were more tolerant than the western ones. These observations inform the basis for the selection of resilient genotypes to be used in breeding and reforestation programs.