Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. The disease is most preferably managed by developing and growing cultivars with high-level, durable resistance. To achieve high-level and long-lasting resistance, we developed a wheat line, RIL-Yr64/Yr15, by pyramiding Yr64 and Yr15, both on the chromosome 1BS and providing high resistance to all tested Pst races. To validate RIL-Yr64/Yr15 possessing both genes, we crossed it to Avocet S (AvS). The F4 RILs from this cross were phenotyped with Pst races under controlled greenhouse conditions and also under natural Pst infection in the field. The population was genotyped with SSR markers previously reported to be linked to the resistance gene loci and with additional SSR and SNP-KASP markers along chromosome 1B. Both phenotype and genotype data confirmed the copresence of Yr64 and Yr15 in RIL-Yr64/Yr15, and the high-resolution linkage map dissected the chromosomal regions and traced their origins. New lines possessing these genes were selected from the F5 population of cross AvS × RIL-Yr64/Yr15 by marker-assisted selection. These lines with the two highly effective genes should be more useful than individual gene lines for developing high-level, durable resistant wheat cultivars.