Instantaneous water use efficiency (WUEi) is a measure made at the leaf scale, which can be used as a criterion for estimating WUE in breeding programs. To study the WUEi in different almond (Prunus dulcis) genotypes, we measured stomatal conductance (g S), assimilation rate (A), transpiration, internal concentration of CO2 (C i), and leaf hydraulic conductance normalized to leaf area in five mixed crosses of almond trees. For all measured parameters we observed the most significant differences between ‘Johnston’ × ‘Lauranne’ and ‘Nonpareil’ × ‘Lauranne’. Nevertheless, ‘Carmel’ × ‘Tarraco’ showed the highest WUEi among the five crosses. The significant correlations among g S, A, and C i indicated that A was probably limited by both stomatal and non-stomatal parameters that might be affected by genotype variations. In another experiment, we selected three cultivars of a new set of almond cultivars (Nonpareil, Carmel, and Masbovera) in four replicates for measuring g S at field capacity. Meanwhile, using a cryo-scanning electron microscopic (SEM) method, we prepared some images from the internal structures of leaves collected from the same cultivars of almond trees. Results showed that ‘Masbovera’ leaves, in which post-venous hydraulic distance (Dm) was higher compared with ‘Carmel’ and ‘Nonpareil’, represented significantly lower values of g S rather than the two other cultivars. Comparing mesophyll anatomy and g S between these cultivars demonstrated that Dm and the density of mesophyll cells might indirectly affect g S in almond leaves. In conclusion, our study demonstrated that water relations, WUEi, and leaf anatomy in almond trees differed among genotypes.