AbstractGrain yield is a highly polygenic trait determined by the number of grains per unit area, as well as by grain weight. In wheat, grain number and grain weight are usually negatively correlated. Yet, the genetic basis underlying trade-off between the two is mostly unknown. Here, we fine-mapped a grain weight QTL using wild emmer introgressions in a durum wheat background, and showed that grain weight is associated with theGNI-A1gene, a regulator of floret fertility. In-depth characterization of grain number and grain weight indicated that suppression of distal florets by the wild emmerGNI-A1allele increase weight of proximal grains in basal and central spikelets due to alteration in assimilate distribution. Re-sequencing ofGNI-A1in tetraploid wheat demonstrated the rich allelic repertoire of the wild emmer gene pool, including a rare allele which was present in two gene copies and contained a non-synonymous mutation in the C-terminus of the protein. Using an F2population generated from a cross between wild emmer accessions Zavitan, which carries the rare allele, and TTD140, we demonstrated that this unique polymorphism is associated with grain weight, independent of grain number. Moreover, we showed, for the first time, thatGNI-A1proteins are transcriptional activators and that selection in domesticated wheat targeted compromised activity of the protein. Our finding expand the knowledge of the genetic basis underlying trade-off between key yield components and may contribute to breeding efforts for enhanced grain yield.