Stripe rust is a major disease constraint of wheat production worldwide. Resistance to stripe rust was analyzed using 131 F6 recombinant inbred lines (RILs) derived from a cross between synthetic derived wheat line Soru#1 and wheat cultivar Naxos. The phenotype was evaluated in Mexico and Norway at both seedling and adult plant stages. Linkage groups were constructed based on 90K single-nucleotide polymorphism (SNP), sequence-tagged site, and simple sequence repeat markers. Two major resistance loci conferred by Soru#1 were detected and located on chromosomes 1BL and 4DS. The 1BL quantitative trait loci explained 15.8 to 40.2 and 51.1% of the phenotypic variation at adult plant and seedling stages, respectively. This locus was identified as Yr24/Yr26 based on the flanking markers and infection types. Locus 4DS was flanked by molecular markers D_GB5Y7FA02JMPQ0_238 and BS00108770_51. It explained 8.4 to 27.8 and 5.5% of stripe rust variation at the adult plant and seedling stages, respectively. The 4DS locus may correspond to known resistance gene Yr28 based on the resistance source. All RILs that combine Yr24/Yr26 and Yr28 showed significantly reduced stripe rust severity in all four environments compared with the lines with only one of the genes. SNP marker BS00108770_51 was converted into a breeder-friendly kompetitive allele-specific polymerase chain reaction marker that will be useful to accelerate Yr28 deployment in wheat breeding programs.