In Citrus species chromosome doubling naturally occurs in somatic embryos and doubled diploid plants often show better adaptation to adverse environmental condition. To understand the molecular determinants of stress acclimation, we examined the response to water deficit in diploid (2×VL) and doubled diploid (4×VL) seedlings of Volkamer lemon (Citrus limonia Osb.) assessing the profile of constitutive volatile organic compound (VOC) in control and stressed conditions. Physiological parameters and leaf volatile compound profiles were measured during water deficit and 24 h after rehydration of plants to field capacity. Net photosynthesis and stomatal conductance were reduced in water stressed leaves, with no significant differences between 2×VL and 4×VL plants. Malondialdehyde concentration, a marker of lipid peroxidation of cellular membranes, was significantly more higher in stressed 2×VL leaves than in 4×VL. The blend of constitutive VOC was different in control leaves being oxygenated monoterpenoids more abundant in 2×VL leaves, and monoterpenoids more abundant in 4×VL leaves. Water deficit did not stimulate biosynthesis of terpenoids, whereas accumulation of trans-2 hexenal, a green leaf volatile (GLV) synthesized after membrane denaturation, was observed in stressed leaves of 2×VL leaves, but not in 4×VL leaves. Semiquantitative PCR showed an increase of the expression of HPL, the gene encoding for hydroperoxidase lyase which catalyzes 2-hexenal formation, only in 2×VL plants. The expression of the putative dehydration transcription factor DREB2A was also observed only in 2×VL water stressed plants. This work shows that level of ploidy may alter constitutive content of GLV by Citrus, therefore likely affecting plants capacity of protection and interaction with other organisms. Whereas diploid and double diploid plants showed similar physiological responses to water deficit, a biochemical marker indicated that membranes of double diploid leaves were more resistan