Americano 44 is a landrace derived wheat cultivar from Uruguay that has had long lasting resistance to the leaf rust pathogen Puccinia triticina. A population of 92 recombinant inbred lines derived from the cross ‘Thatcher*3/Americano 44d’ was evaluated for segregation of adult plant leaf rust resistance in four field plot tests and two greenhouse tests. A genetic map was constructed with 381 Diversity Array Technology markers, five simple sequence repeat markers, and seven kompetitive allele specific PCR markers. Significant effects for reduction of leaf rust severity were found for quantitative trait loci (QTL) on chromosomes 3AS, 3DS and 6DS. Individually the 3AS and 3DS regions did not decrease leaf rust severity in any of the tests compared to the susceptible parent, yet strongly interacted when present in the same genotype to decrease leaf rust severity equal to the resistant parent. Genotypes with only the 6DS region had significantly lower leaf rust severity compared to the susceptible parent in three of the field plot tests. The 6DS QTL also interacted with the 3DS QTL to decrease leaf rust severity. Major QTLs on chromosome 3AS and 3DS are both located in the distal region and may be encoded by homoeoalleles.