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. Improved cultivars often lose their resistance due to occurrence of new virulent races which overcome the genes and make the cultivars out of production. Therefore, identification of new sources of resistance genes helps in battling yellow rust and maximizes wheat production in Ethiopia. In this study, 300 durum wheat lines (landraces & cultivars) were screened with three virulent isolates (Pst_Is1, Pst_Is4 and Pst_Is8) for seedling resistance using Infection Type (IT) scoring method. The lines were also screened with 16 KASP-based SNP markers linked to 7 Yr genes already identified in various studies. Highly resistant infection type (IT: 0-3) to Pst_Is1, Pst_Is4, and Pst_Is8 was exhibited by 59.3%; 67.3%; and 46.3% of the lines, respectively. 124 lines constantly exhibited high level of resistance to all three isolates. The majority (96.8%) of the resistant lines are landraces while four (3.2%) are commercial cultivars (Cocorit/71, Yerer, Obsa and Dire). In the molecular screening 12 of the markers gave clear amplifications in the controls and the tested lines. Yr7, Yr15 and YrSp were detected in 81.7%, 88.3% and 0.7% of the lines respectively while Yr1, Yr17 and Yr36 were not detected. Detection frequency was higher in landraces (58.7%) than in cultivars (32.8%). Gene combinations frequency was the highest (72.7%) for Yr7+Yr15 followed by Yr15+YrSp (0.3%). Overall, this study has resulted in detection of genes Yr15 and YrSp, which are potential candidates for marker assisted breeding for Pst resistance in wheat. Besides, it has shown that resistant source identification and detection of genes can be facilitated through combined application of phenotyping and molecular screening.