Wild relatives of wheat serve as an extraordinary source of variability for breeding programs due to their capabilities to respond to various environmental stresses. Here, we investigated some species possessing a D genome (T. aestivum, Ae. tauschii, Ae. crassa and Ae. cylindrica) in terms of relative water content (RWC), stomatal conductance (Gs), relative chlorophyll content, initial fluorescence (Fo), maximum quantum yield of PSII (Fv/Fm), maximum primary yield of PSII photochemistry (Fv/Fo), as well as shoot fresh and dry biomasses under control and water deficit conditions. Our results revealed that water deficit negatively affected all traits; shoot fresh weight, Gs and RWC showed the highest reduction compared to the control condition. Principal component analysis (PCA) identified two PCs that accounted for 53.36% of the total variation in the water deficit conditions. Correlation analysis and PCA-based biplots showed that stress tolerance index (STI) is significantly associated with Fv/Fm and Fv/Fo under water stress conditions, suggesting that these are the best parameters to evaluate when screening for tolerant samples at the seedling stage. We identified 19 accessions from Ae. crassa and one from Ae. tauschii as the most tolerant samples. In conclusion, Ae. crassa might provide an ideal genetic resource for drought-tolerant wheat breeds.