Stripe (Yellow) rust caused by Puccinia striiformis f.sp. tritici (Pst) is one of the most devastating diseases of wheat in the highlands of Ethiopia. Identification of resistant genes helps in battling yellow rust and maximize wheat production. Virulence phenotyping was carried out on ten Pst isolates originally collected from three testing sites (Meraro, Kulumsa and Chefe-Donsa) using a set of 35 stripe rust differential lines corresponding to 19 resistance genes: Yr1, Yr2, Yr3, Yr4, Yr5, Yr6, Yr7, Yr8, Yr9, Yr10, Yr15, Yr17, Yr24, Yr25, Yr27, Yr32, YrSp, YrAvS and Yr Amb. Three isolates (Pst_Is1, Pst_Is4 and Pst_Is8) that combined virulence to all but Yr5, Yr15 and YrSp were identified and used to test 300 durum wheat lines for seedling resistance in the greenhouse at Kulumsa research center. The result showed that, 56.3%; 63.3%; and 46.3%, of the lines exhibited highly resistant infection type (0 < IT < 3) in response to Pst_Is1, Pst_Is4, and Pst_Is8 respectively. Percentage of resistant lines ranged from 43.6 - 64.1, 38.7 - 61.5, and 12.8 - 17.9 within landraces, selections and cultivars respectively. Presence of seven Yr genes was investigated with haplotype analysis of 16 linked KASP-based SNP markers on the same set of durum lines including Yr-differentials and YrAvS as positive and negative controls respectively. Twelve of the markers gave clear co-segregation of the tested lines between positive and negative controls of the Yr genes. On the average, 80.7% and 56.2% of the lines clearly co-segregated with the positive controls of Yr7 and Yr15 respectively. Only two lines (0.7%) co-segregated for YrSp while 98.7% co-segregated (including the negative control) for Yr5 suggesting the need for retesting them with perfect diagnostic markers. No evidence was found for the presence of Yr1, Yr17 and Yr36. Average cosegregation of lines with Yr7, Yr15, Yr5 and YrSp positive controls was 61.4%, 65.4% and 41.8% within landraces, selections and cultivars respectively. In conclusion, in both phenotypic and molecular screening, landraces and selection have shown to be better sources for Pst resistance over released cultivars. Besides, the tested lines carry haplotypes at least similar if not identical to the respective Yr genes. Our result gives evidence that in Ethiopia, Yr15 can be used for marker assisted breeding while Yr5 and YrSp can still be good candidates provided that they are reconfirmed with perfect diagnostic markers.