Disease resistance breeding of wheat using molecular markers aims to efficiently identify and incorporate key resistance genes against stripe rust into elite wheat cultivars. This approach enhances the precision and speed of breeding programs to develop durable, resistant wheat varieties capable of withstanding Puccinia striiformis f. sp. tritici (Pst) infections, ultimately contributing to sustainable wheat production and global food security. A total of 174 bread wheat genotypes, comprising 94 Turkish cultivars and 80 exotic landraces obtained from the USDA, were screened using gene-specific markers for the stripe rust resistance genes Yr15 and Yr5. The Yr15 resistance gene was detected in 31 genotypes, including 22 Turkish cultivars and nine USDA landraces. The Yr5 resistance gene was not found in any of the tested germplasm, but the susceptibility gene was identified in 77 genotypes. Turkish cultivars showed higher susceptibility to stripe rust, with 46 out of 94 genotypes vulnerable, compared to 25 out of 80 USDA genotypes. These findings highlight the urgent need to integrate Yr15 and other resistance genes into wheat breeding programs to enhance crop resilience against evolving Pst isolates, thereby ensuring sustainable wheat production and contributing to global food security.