Carbon isotope discrimination (Δ) has been proposed as physiological criterion to select C3 crops for yield and water use efficiency. The relationships between carbon isotope discrimination (Δ), water use efficiency for grain and biomass production (WUEG and WUEB, respectively) and plant and leaf traits were examined in 20 Iranian wheat genotypes including einkorn wheat (Triticum monococcum L. subsp. monococcum) accessions, durum wheat (T. turgidum L. subsp. durum (Desf.) Husn.) landraces and bread wheat (T. aestivum L. subsp. aestivum) landraces and improved cultivars, grown in pots under well-watered conditions. Carbon isotope discrimination was higher in diploid than in hexaploid and tetraploid wheats and was negatively associated with grain yield across species as well as within bread wheat. It was also positively correlated to stomatal frequency. The highest WUEG and grain yield were noted in bread wheat and the lowest in einkorn wheat. Einkorn and bread wheat had higher WUEB and biomass than durum wheat. WUEG and WUEB were significantly negatively associated to Δ across species as well as within bread and durum wheat. The variation for WUEG was mainly driven by the variation for harvest index across species and by the variation for Δ within species. The quantity of water extracted by the crop, that was closely correlated to root mass, poorly influenced WUEG. Environmental conditions and genetic variation for water use efficiency related traits appear to highly determine the relationships between WUEG and its different components (water consumed, transpiration efficiency and carbon partitioning).