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Intensive breeding and replacement of traditional landraces by modern cultivars led to the narrowing of genetic variation in cultivated wheat. The most sustainable method for wheat improvement is utilization of genetic diversity from wheat wild relatives such as Aegilops speltoides that has a diversity of genes for resistance to leaf rust (LR). A high pairing-inducing Ae. speltoides strain collected from Israel was introgressed into T. turgidum subsp. durum var. landrace Nursi. The F1 plants were treated with colchicine to induce chromosome doubling. The resulting hexaploid plants were crossed to bread wheat cv. Beit-Lehem and F3 plants were backcrossed three times to bread wheat cv. Barnir. Each generation was selected for LR resistance to P. triticina isolate #1010 and five resistant wheat-Ae. speltoides introgression lines (ILs) designated DK1 to DK5 were selected. These Ae. speltoides ILs were genotyped using the 90K Infinium SNP assay and most of the polymorphic markers were mapped to chromosome 1B suggesting that the Ae. speltoides introgressions encompass most of this chromosome. To test if the newly identified gene is identical to Lr51, that was also introgressed from Ae. speltoides to chromosome 1B of bread wheat, the DK ILs were genotyped with the molecular marker AGA7 that was shown to be linked to Lr51. The Ae. speltoides AGA7 allele was absent in the DK ILs suggesting that these genotypes are not carrying the Lr51 introgression. Moreover, we performed an allelism test. Spring wheat cv. Kern harboring resistance gene Lr51 was crossed with DK2 and an F2 segregation ratio of 15R:1S was obtained, indicating that the resistance was conditioned by two independent dominant genes. Overall, our results suggest that DK2 carries a new leaf rust resistance gene from Ae. speltoides and this gene has potential for wheat improvement.