Stomatal traits are determinants of plant water relations and may differ among cultivars within a given species. These features have been rarely studied in tree crops, such as pistachio (Pistacia vera L.), the cultivation of which is expanding in several Mediterranean countries due to its economics revenues. Therefore, this study aims at characterizing several stomata features (length, width, surface, and density) in three pistachio cultivars (Golden Hills, Lost Hills, and Kerman) growing in Central Spain. In addition, the relationships between these traits and gas exchange and stem water potential (Ψs) measurements were explored over the 2025 growing season. Kerman trees had more negative Ψs values than the other cultivars when atmospheric demand was high, which translated into lower stomatal conductance and net photosynthesis values. This coincided with lower stomatal density and specific leaf area in Kerman. However, stomata were bigger in Kerman than in the other cultivars. When compared over the course of the day, stomatal conductance in the abaxial leaf surface was, approximately, 70% greater than that observed on the adaxial side of the leaves in the three cultivars. These findings provide relevant insights for understanding the regulation of water relations in this species, which may serve for planning new plantations.