Wheat is one of the most important cereal crops of Ethiopia. Stem rust caused by Puccinia graminis f. sp. tritici is amongst the biotic factors which can cause up to 100% yield loss if susceptible cultivar grown and epidemic occurs. The highland of Ethiopia is considered as a hot spot for the development of stem rust diversity. This study was carried out to determine virulence diversity and race distribution of P. graminis f. sp. tritici in Ethiopia. Eighty (80) wheat stem rust samples were collected in 2013 cropping season from Oromia, Amhara and Tigray region. Of the samples collected, 66 were analyzed on to the 20 stem rust differential lines. A total of 9 races were identified, which includes TTKSK, TTKTF, TTKTK, JRCQC, TKTTF, TTKSC, TRTTF, SRKSC and RRKSF. Race TTKSK was dominant and widely distributed in the Oromia and Amhara regions with 52% frequency; it was not isolated in Tigray region. The most virulent and new race, TKTTF which causes localized stem rust epidemic in Bale and Arsi was predominantly distributed in Oromia region with 36.4% frequency value. Most of the genes possessed by the differentials were ineffective against one or more of the tested isolates. Only stem rust resistance gene 24 was found to confer resistance to all of the races isolated in this study. This gene could be used in combination with other genes through gene pyramiding in breeding for resistance to stem rust in Ethiopia.

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