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Aegilops tauschii, the progenitor of the wheat D genome, contains extensive diversity for biotic and abiotic resistance. Lr21 is a leaf rust resistance gene, which did not enter the initial gene flow from Ae. tauschii into hexaploid wheat due to restrictive hybridization events. Here, we used population genetics and high-resolution comparative genomics to study evolutionary and functional divergence of Lr21 in diploid and hexaploid wheats. Population genetics identified the original Lr21, lr21-1 and lr21-2 alleles and their evolutionary