Stripe rust caused by the pathogen Puccinia striiformis f. sp. tritici (Pst) is a major threat for wheat, resulting in low yield and grain quality loss in many countries. Genetic resistance is a prevalent method to combat the disease. Mapping the resistant loci and their association with traits is highly exploited in this era. A panel of 465 Pakistani spring wheat genotypes were evaluated for their phenotypic response to stripe rust at the seedling and adult plant stages. A total of 765 single nucleotide polymorphism (SNP) markers were applied on 465 wheat genotypes to evaluate their stripe rust response against nine races during the seedling test and in three locations for the field test. Currently, twenty SNPs dispersed on twelve chromosomal regions (1A, 1B, 1D, 2A, 2B, 4A, 4B, 5B, 6A, 6B, 6D and 7B) have been identified that were associated with rust race-specific resistance at the seedling stage. Thirty SNPs dispersed on eighteen chromosomal regions (1A, 1B, 1D, 2A, 2B, 2D, 3A, 3B, 3D, 4B, 5A, 5B, 6A, 6B, 6D, 7A, 7B and 7D) are associated with adult plant resistance. SNP loci IWB3662 was linked with all three Pakistani races, and likewise IWA2344 and IWA4096 were found to be linked with three different USA races. The present research findings can be applied by wheat breeders to increase their resistant capability and yield potential of their cultivars, through marker-assisted selection.