Each year, significant global wheat yield loss occurs due to diseases that affect yield quantity or quality. Breeding for resistance has been the best economic and environmentally safe approach to control wheat diseases, however many disease resistance (R) genes succumbed to the pathogens and are no longer effective. Hence, new sources of resistance are necessary to boost the wheat gene pool. The main source for such genes are species of wheat wild relatives in the secondary gene pool that contain an unexploited reservoir of novel R genes. Sharon goatgrass (Aegilops sharonensis Eig) is a wild diploid relative of wheat (genome SshSsh). It is native to the coastal plain of Israel, growing mostly on stabilized dunes, and is highly resistant to rust pathogens. Previously, we introgressed a leaf and stripe rust resistance locus from Ae. sharonensis into bread wheat using chromosome engineering (Millet et al., 2014). We mapped the alien region to the short arm of chromosome six using genotyping by sequencing, identified SNPs, and used them to generate diagnostic markers (Khazan et al., 2020). Here, we report on the isolation of a candidate NLR-type R gene from the wheat introgression lines by a combination of mutational genomics, RNA-seq and chromosome sorting and sequencing.