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Wheat stem rust, caused by the fungus <i>Puccinia graminis</i> f. sp. <i>tritici</i> (<i>Pgt</i>), poses a substantial threat to global wheat production. Utilizing stem rust resistance (<i>Sr</i>) genes represents an economically viable, effective, and environmentally friendly approach to disease control. In this study, gene postulation, molecular testing, and pedigree analysis were used to identify the presence of <i>Sr</i> genes in 45 wheat cultivars. In addition, the resistance of these cultivars was evaluated against two predominant <i>Pgt</i> races, 34MRGQM and 21C3CTHTM, at the adult-plant stage during 2021–2022. The results identify seven <i>Sr</i> genes (<i>Sr31</i>, <i>Sr38</i>, <i>Sr30</i>, <i>SrTmp</i>, <i>Sr22</i>, <i>Sr19</i>, and <i>Sr5</i>) within 35 wheat cultivars. Among these, 23 cultivars contained <i>Sr31</i>, whereas <i>Sr5</i> and <i>SrTmp</i> were present in four cultivars each. Han 5316, Shimai 15, Shiyou 20, and Kenong 1006 exhibited the presence of <i>Sr19</i>, <i>Sr22</i>, <i>Sr30</i>, and <i>Sr38</i>, respectively. Molecular studies confirmed the absence of <i>Sr25</i> and <i>Sr26</i> in any of the wheat cultivars. During field evaluation, 37 (82.2%) and 39 (86.7%) wheat cultivars demonstrated resistance to races 34MRGQM and 21C3CTHTM, respectively. Moreover, 33 wheat cultivars (73.3%) exhibited resistance to all the tested races. These study findings will significantly contribute to future research in wheat pre-breeding and abiotic stress tolerance.