The tools for unraveling the mechanisms of disease resistance in cereal crops are currently limited due to their large repeat-rich genomes and the lack of genetic recombination or cultivar (cv)-specific sequence information. We cloned the first leaf rust resistance gene Rph1 from cultivated barley using “MutChromSeq,” a recently developed molecular genomics approach for the rapid cloning of genes in plants. Flow-sorted 2H chromosomes from Sudan (wild type) and six sodium azide mutants were sequenced. Sequence analysis revealed a single gene candidate encoding a coiled-coil nucleotide binding site Leucine-rich repeat (NLR) receptor protein. The gene Rph1was mapped to the short arm of chromosome 2H in a physical region of 1.3 megabases. We determined the molecular basis for Rph1-mediated resistance in cultivated barley enabling varietal improvement through diagnostic marker design, gene editing, and gene stacking technologies.