Following emergence of Ug99, the new virulent race of Puccinia graminis f. sp. tritici in Africa, a global effort for identification and utilization of new sources of Ug99-resistant germplasm has been undertaken. In this study, we conducted replicated experiments to evaluate the resistance of Iranian wheat germplasm to the TTKSK lineage of the Ug99 race of P. graminis f. sp. tritici. We also evaluated for presence of stem rust resistance genes (i.e., Sr2, Sr24, Sr26, Sr38, Sr39, Sr31, and Sr1RSAmigo) in wheat cultivars and breeding lines widely cultivated in Iran. Our phenotyping data revealed high levels of susceptibility to Ug99 in Iranian bread wheat germplasm. Our genotyping data revealed that Iranian cultivars do not carry Sr24, Sr26, or Sr1RSAmigo. Only a few salt-tolerant cultivars and breeding lines tested positively for Sr2, Sr31, Sr38, or Sr39 markers. In conclusion, the genetic basis for resistance to Ug99 in Iranian wheat cultivars was found to be vulnerable. Acquiring knowledge about existing resistance genes and haplotypes in wheat cultivars and breeding lines will help breeders, cereal pathologists, and policy makers to select and pyramid effective stem rust resistance genes.