The ear, together with the flag leaf, is believed to play a major role as a source of assimilates during grain filling in C₃ cereals. However, the intrusive nature of most of the available methodologies prevents reaching conclusive results in this regard. This study compares the carbon isotope composition (δ¹³C) in its natural abundance in the water-soluble fractions of the flag leaf blade and the ear with the δ¹³C of mature kernels to assess the relative contribution of both organs to grain filling in durum wheat (Triticum turgidum L. var. durum). The relative contribution of the ear was higher in landraces compared to modern cultivars, as well as in response to nitrogen fertilization and water stress. Such genotypic and environmentally driven differences were associated with changes in harvest index (HI), with the relative contribution of the ear being negatively associated with HI. In the case of the genotypic differences, the lower relative contribution of the ear in modern cultivars compared with landraces is probably associated with the appearance in the former of a certain amount of source limitation driven by a higher HI. In fact, the relative contribution of the ear was far more responsive to changes in HI in modern cultivars compared with landraces.