Stem rust is a devastating disease of cereal crops worldwide. In barley (Hordeum vulgare), the disease is caused by two pathogens: Puccinia graminis f. sp. secalis (Pgs) and Puccinia graminis f. sp. tritici (Pgt). In North America, the stem rust resistance gene Rpg1 has protected barley from losses for more 60 years; however, widely virulent Pgt races from Africa in the Ug99 group threaten the crop. To identify novel quantitative trait loci (QTL) for stem rust resistance, bi-parental and association mapping studies were undertaken in the Barley iCore Collection (BCC) held by the USDA National Small Grains Collection. Association mapping studies of the BCC were conducted for seedling resistance to Pgt race TTKSK (Ug99 group) in the greenhouse and adult plant resistance (APR) to Pgt TTKSK composite in Njoro, Kenya and Pgt race QCCJB in St. Paul, MN. A major effect QTL (Rpg-qtl-5H-11_11355) for APR in all locations was identified on chromosome 5H. This QTL represents a unique locus for APR and has been confirmed in other studies. Subsequently, 290 of the most resistant BCC accessions, the BCC Selects (BCCS), were screened for resistance Pgt races MCCFC, QCCJB, HKHJC, TTKSK, TTKST, TKTTF, and TRTTF, and also Pgs isolate 92-MN-90. From this investigation, four BCCS accessions were postulated to carry Rpg1, 14 to carry Rpg2, 91 to carry Rpg3, four to carry rpg4/Rpg5, and 59 to carry potentially novel resistance genes. To map the APR genes of Rpg2 and Rpg3 in Hietpas-5 (CIho 7124) and GAW-79 (PI 382313), respectively, two biparental populations were developed with Hiproly (PI 60693), a stem rust susceptible accession. Both populations were phenotyped to the domestic Pgt races of MCCFC, QCCJB, and HKHJC in St. Paul and to Pgt TTKSK composite in Njoro. In the Heitpas-5/Hiproly population, a major effect QTL was identified on chromosome 2H, which is proposed as the location for Rpg2. In the GAW-79/Hiproly population, a major effect QTL was identified on chromosome 5H and is the proposed location for Rpg3. The resistance sources identified and characterized in this study enhance barley breeding programs focused on stem rust resistance.