<p>Stomata serve as a crucial experimental tool for investigating plant responses to environmental changes, with their size and density being key eco-physiological parameters that jointly influence stomatal conductance. Stomatal aperture dynamics are governed by anatomical characteristics, such as pore and guard cell dimensions, which exhibit a strong relationship with the plant’s water status. We examined the trends in stomatal anatomical traits of nine citrus rootstock genotypes under sufficient moisture (control) and drought stress (DS). The predominant occurrence of the ‘Anomocytic’ stomatal complex was observed among the tested genotypes. The trifoliate hybrids, Troyer citrange and X639, exhibited significantly larger stomatal apertures compared to the mono-foliate species, with mean values of 54.77 µm² and 49.58 µm², respectively. The highest stomatal density (485.4&#xa0;mm⁻²) and epidermal cell count were documented in RLC-4 and X639 plants, respectively. DS increased the percentage of closed stomata by 39.61% and reduced the potential conductance index by 37.31%. Additionally, a positive correlation was observed between stomatal pore length and width and physiological parameters, such as net photosynthesis, transpiration rate, membrane stability index (MSI), relative water content (RWC), and leaf count. Heatmap and cluster analysis identified two major sub-clusters in the tested genotypes, with the first sub-cluster comprising variants of rough lemon and the second cluster consisting of Cleopatra mandarin, X639, and Troyer citrange. Based on stomatal responses under DS, X639 and Troyer citrange can be classified as ‘anisohydric,’ whereas the remaining genotypes exhibit an ‘isohydric’ behaviour. This study offers new insight into how stomatal microstructure shapes drought-responsive behaviour in citrus, thereby strengthening the physiological understanding of genotype-specific adaptation strategies.</p> Graphical abstract