The Triticum turgidum ssp. dicoccum (2n = 4x = 28) accession MG5323 showed a useful level of resistance to leaf rust disease. A segregating population of 110 recombinant inbred lines (RILs), derived from a cross between cv Latino (T. turgidum spp. durum), susceptible to leaf rust, and MG5323 was evaluated for reactions of seedlings to two different Puccinia triticina isolates. Genotyping of the RILs was performed with different molecular markers (SSR, EST-SSR and SNP), leading to the construction of a linkage map containing 10,840 loci covering 14 chromosomes, with an average marker density of 0.22 cM/marker. Linkage analysis allowed the identification of three different regions significantly associated with leaf rust resistance, with MG5323 contributing the resistant alleles. A major resistance gene was detected on the short arm of chromosome 1B, explaining a total phenotypic variation ranging from 41.37 to 49.51 %. Two additional minor resistance genes located on chromosome 7B explained a phenotypic variation ranging between 17.77 and 25.81 %. No obvious positional relationships were observed when the map position of the genes was compared with those of other previously identified wheat leaf rust resistance genes, suggesting that new resistance sources to leaf rust were identified in the tetraploid background. A significant positive epistatic effect was detected between quantitative trait loci (QTLs) for each trait, indicating that different QTLs contribute different degrees of resistance. Analysis of the leaf rust responses of the RILs demonstrated that only lines bearing resistant alleles at both loci showed effective leaf rust resistance, indicating that the genes identified behave as complementary genes.