Race TTKSK (Ug99) of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici) is a serious threat to both wheat and barley production worldwide because of its wide virulence on many cultivars and rapid spread from eastern Africa. Line Q21861 is one of the most resistant barleys known to this race. To elucidate the genetics of resistance in this line, we evaluated the Q21861/SM89010 (Q/SM) doubled-haploid population for reaction to race TTKSK at the seedling stage. Segregation for resistance:susceptibility in Q/SM doubled-haploid lines fit a 1:1 ratio (58:71 with chi2=1.31 and P=0.25), indicating that a single gene in Q21861 confers resistance to race TTKSK. In previous studies, a recessive gene (rpg4) and a partially dominant gene (Rpg5) were reported to control resistance to P. graminis f. sp. tritici race QCCJ and P. graminis f. sp. secalis isolate 92-MN-90, respectively, in Q21861. These resistance genes co-segregate with each other in the Q/SM population and were mapped to the long arm of chromosome 5H. Resistance to race TTKSK also co-segregated with resistance to both rusts, indicating that the gene conferring resistance to race TTKSK also lies at the rpg4/Rpg5 locus. This result was confirmed through the molecular analysis of recombinants previously used to characterize loci conferring resistance to race QCCJ and isolate 92-MN-90. The 70-kb region contains Rpg5 (a nucleotide-binding site leucine-rich repeat serine/threonine-protein kinase gene), rpg4 (an actin depolymerizing factor-like gene), and two other genes of unidentified function. Research is underway to resolve which of the genes are required for conferring resistance to race TTKSK. Regardless, the simple inheritance should make Q21861 a valuable source of TTKSK resistance in barley breeding programs.