Stripe rust caused by Puccinia striiformis f.sp. tritici (Pst) is threatening wheat production in Ethiopia. Wheat varieties succumb to new Pst race(s) soon after their release from research centers. This stusy aimed to determine prevalent Pst races and identify resistance sources in the Ethiopian wheat landraces. A total of 44 Pst samples were collected for race analysis from the Amhara and Oromia regions. The field test was conducted at Kulumsa and Meraro, while the seedling tests were conducted at Kulumsa in a greenhouse. Seven Pst phenotypic races were identified from 44 samples, and PstS11 was the most frequent (36.4%), whereas three distinct Pst races were identified from 24 samples by SSR genotyping. Of the 103 wheat landraces (69 bread and 34 durum), 57 exhibited resistance across both locations and seasons. The 57 Ethiopian wheat landraces that showed field resistance were further exposed to three Pst races at the seedling stage, and 32 Ethiopian wheat landraces exhibited seedling resistance to all races. The resistance in these materials will be genotyped and used in the wheat breeding program.