Abstract Osmotic adjustment is almost ubiquitous as a mechanism of response to drought in many forest species. Recognized as an important mechanism of increasing turgor under water stress, the metabolic basis for osmotic adjustment has been described in only a few species. We set an experiment with four species of the genus Quercus ranked according to drought tolerance and leaf habit from evergreen to broad-leaved deciduous. A cycle of watering deprivation was imposed on seedlings, resulting in well-watered (WW) and water-stressed (WS) treatments, and their water relations were assessed from pressure–volume curves. Leaf predawn water potential (Ψpd) significantly decreased in WS seedlings, which was followed by a drop in leaf osmotic potential at full turgor (Ψπ100). The lowest values of Ψπ100 followed the ranking of decreasing drought tolerance: Quercus ilex L. < Quercus faginea Lam. < Quercus pyrenaica Willd. < Quercus petraea Matt. Liebl. The leaf osmotic potential at the turgor loss point (ΨTLP) followed the same pattern as Ψπ100 across species and treatments. The pool of carbohydrates, some organic acids and cyclitols were the main osmolytes explaining osmotic potential across species, likewise to the osmotic adjustment assessed from the decrease in leaf Ψπ100 between WW and WS seedlings. Amino acids were very responsive to WS, particularly γ-aminobutyric acid in Q. pyrenaica, but made a relatively minor contribution to osmotic potential compared with other groups of compounds. In contrast, the cyclitol proto-quercitol made a prominent contribution to the changes in osmotic potential regardless of watering treatment or species. However, different metabolites, such as quinic acid, played a more important role in osmotic adjustment in Q. ilex, distinguishing it from the other species studied. In conclusion, while osmotic adjustment was present in all four Quercus species, the molecular processes underpinning this response differed according to their phylogenetic history and specific ecology.