Breeding for durable leaf rust resistance is a priority for our breeding programs; however, the availability of new resistance genes is a limiting factor. Two spring wheat populations totaling 186 lines derived from three resistant donors and two Uruguayan susceptible cultivars were used to detect genomic regions associated with seedling and field resistance to LR in Uruguay. AUDPC were recorded in three environments in the 2012 and 2013 cropping seasons, and seedling responses were determined using three Puccinia triticina races. The lines were also genotyped using GBS. A total of 5,222 SNP markers were used for genome-wide association analysis. Molecular markers were used to genotype APR genes Lr34 and Lr68. We identified 43 SNP markers significantly associated with seedling resistance and 19 for field resistance on chromosomes 1A, 1B, 1D, 2B, 2D, 3A, 4A, 5B, 6B, 7A, 7B and 7D. We confirmed the presence of Lr10 and Lr16 in seedling tests and Lr34 and Lr68 in field tests. Novel genomic regions were identified on chromosomes 4A associated with APR, and 5B associated with seedling resistance. These new resistance genes will be useful in breeding for durable LR resistance.