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Stripe rust, caused by Puccinia striiformis f. sp. tritici , is one of the most devastating wheat diseases in Ethiopia. To study virulence genetics of the pathogen, 117 progeny isolates were produced through sexual reproduction of an Ethiopian isolate of the stripe rust pathogen on Berberis holstii plants under controlled conditions. The parental and progeny isolates were characterized by phenotyping on wheat lines carrying single Yr genes for resistance and genotyped using 10 polymorphic simple sequence repeated (SSR) markers. The progeny isolates were classified into 37 virulence phenotypes and 75 multilocus genotypes. The parental isolate and progeny isolates were all avirulent to resistance genes Yr5 , Yr10 , Yr15 , Yr24 , Yr32 , YrTr1 , YrSP , and Yr76 but virulent to Yr1 and Yr2 , indicating that the parental isolate was homozygous avirulent or homozygous virulent at these loci. The progeny isolates segregated for virulence to 12 Yr genes. Virulence phenotypes to Yr6 , Yr28 , Yr43 , and Yr44 were controlled by a single dominant gene; those to Yr7 , Yr9 , Yr17 , Yr27 , Yr25 , Yr31 , and YrExp2 were each controlled by two dominant genes; and the virulence phenotype to Yr8 was controlled by two complementary dominant genes. A linkage map was constructed with seven SSR markers, and 16 virulence loci corresponding to 11 Yr resistance genes were mapped with some loci linked to each other. These results are useful in understanding host-pathogen interactions and selecting resistance genes to develop wheat cultivars with highly effective resistance to stripe rust.