Stripe rust (or yellow rust), caused by Puccinia striiformis f. sp. tritici, is the most destructive disease of wheat worldwide, especially in the United States. To identify loci for effective stripe rust resistance in U.S. wheat, a genome-wide association study (GWAS) was conducted using two panels of 616 spring wheat and 857 winter wheat cultivars and breeding lines. The accessions in these panels were phenotyped for stripe rust response in the greenhouse at seedling stage with multiple predominant and highly virulent races of the pathogen and at adult-plant stage in multiple field environments. The accessions were genotyped using a genotyping by multiplexed sequencing (GMS) platform. In addition, molecular markers of previously reported resistance genes or quantitative trait loci (QTL) were also used to genotype the panels. The spring and winter panels were grouped into sub-populations based on principal component (PC) analysis. In the spring panel, a total of 37 loci on 15 chromosomes (1A, 1B, 2A, 2B, 2D, 3B, 3D, 4A, 4B, 5A, 5B, 6B, 6D, 7A, and 7D) for resistance to stripe rust were detected including 10 potentially new QTL. In the winter panel, GWAS analysis identified 34 loci also on 15 chromosomes (1A, 1B, 1D, 2A, 2B, 2D, 3A, 4A, 4B, 5A, 5B, 5D, 6A, 7A, and 7B) significantly associated to stripe rust resistance. Comparison of the loci identified in the winter panel with previously mapped loci identified at least four new loci for stripe rust resistance. Of the 15 previously reported genes tested with molecular markers, 13 were present in the US winter wheat cultivars and breeding lines, and some of them were also identified in the GWAS analysis. The frequencies of the resistance genes or QTL in various regional nurseries were determined, indicating different use intensities of these genes or QTL in breeding programs in different regions. The resistance loci and the information on their markers, effectiveness, and distributions should be useful for improving stripe rust resistance in wheat cultivars.