Virulence surveys of Puccinia hordei of barley were conducted in the main and off crop seasons of 2010/11 and 2011/12 in West Shewa, Wellega (western part of the country) and Arsi (central part of the country) zones of Oromiya region, Ethiopia to determine the virulence spectrum of the pathogen, and identify the effective resistance genes to the pathotypes. In the two crop seasons, 56 leaf (brown) rust samples in the main and 32 in the offseason were collected. From each barley field, single pustule descent spores were multiplied and inoculated onto the seedlings of 12 leaf rust differentials carrying Rph1 - Rph12 genes to designate the pathotypes. A total of 88 leaf rust isolates were processed and based on infection phenotype on the resistance genes, 7 pathotypes (ETPh6631, ETPh6611, ETPh6671, ETPh7671, ETPh7631, ETPh7611 and ETPh7651) were identified. The most frequently isolated pathotype was ETPh6631 with 43.2% followed by ETPh6611 with 19%. Moreover, virulence spectrum of P. hordei pathotypes identified in this study was diverse. Resistance genes with Rph1 (Sudan), Rph4 (Gold), Rph8 (Egypt4), Rph9 (Hor 2596), Rph11 (Clipper BC68) and Rph12 (Triumph) were non-effective to all pathotypes identified whereas genes Rph5 (Magnif), Rph6 (Bolivia) and Rph10 (Clipper BC 8) were effective to 26.1, 73.9 and 78.4% of the isolates, respectively. Virulence against Rph2 (Peruvian), Rph3 (Estate) and Rph7 (Cebada Capa) was absent. Therefore, the effective major genes to the existing leaf rust populations could be utilized as sources of resistance in the barley breeding program.