Race TTKSK of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici) threatens the production of wheat and barley worldwide because of its broad-spectrum virulence on many widely grown cultivars. Sources of resistance against race TTKSK were recently identified in several barley landraces (Hordeum vulgare subsp. vulgare) and wild barley accessions (H. vulgare subsp. spontaneum). The objectives of this study were to characterize the inheritance of resistance to wheat stem rust race TTKSK in four barley landraces (Hv501, Hv545, Hv602, and Hv612) and two wild barley (WBDC213 and WBDC345) accessions, map the resistance genes, and determine the allelic relationships among the genes in these accessions and the previously described rpg4/Rpg5 locus. Resistant accessions were crossed with the susceptible cv. Steptoe and resulting F3 populations were evaluated for resistance to race TTKSK at the seedling stage. Segregation of F3 families in populations involving the resistance sources of Hv501, Hv545, Hv612, WBDC213, and WBDC345 fit a 1:2:1 ratio for homozygous resistant (HR)/segregating (SEG)/homozygous susceptible (HS) progenies (with χ2 = 2.27 to 5.87 and P = 0.053 to 0.321), indicating that a single gene confers resistance to race TTKSK. Segregation of F3 families in cross Steptoe/Hv602 did not fit a 1:2:1 ratio (HR/SEG/HS of 20:47:43 with χ22 = 11.95 and P = 0.003), indicating that more than one gene is involved in imparting resistance to race TTKSK. Bulked segregant analysis using >1,500 single-nucleotide polymorphism markers positioned a resistance locus in all six populations on chromosome 5HL in very close proximity to the known location of the rpg4/Rpg5 complex locus. Allelism tests were conducted by making crosses among resistant accessions Hv501, Hv545, and Hv612 and also Q21861 with the rpg4/Rpg5 complex. No segregation was observed in F2 families inoculated with race TTKSK, demonstrating that all Hv lines carry the same allele for resistance and that it resides at or very near the rpg4/Rpg5 locus. Phenotype evaluations of the six barley accessions with wheat stem rust race QCCJ revealed resistant infection types (ITs) at a low incubation temperature and susceptible ITs at a high incubation temperature, similar to Q21861, which carries the temperature-sensitive gene rpg4. The accessions also exhibited low ITs against the rye stem rust isolate 92-MN-90, suggesting that they also carry Rpg5. This result was confirmed through molecular analysis, which revealed that all six barley accessions contain the serine threonine protein kinase domain that confers Rpg5 resistance. These results indicate that cultivated barley is extremely vulnerable to African stem rust races such as TTKSK because even these diverse selections of landrace and wild barley accessions carry only one locus for resistance.