The Volga region is one of the main grain-producing regions of Russia. Wheat stem rust caused by Puccinia graminis f. sp. tritici is among the most destructive fungal diseases of wheat. Recently, its harmfulness has increased in the Volga region. In this regard, an analysis of the resistance and diversity of the Sr genes in the Russian wheat cultivars is necessary. In this work, 126 wheat cultivars (including 23 durum wheat cultivars and 103 bread wheat cultivars) approved for use in the Volga region were evaluated for their resistance to two samples of P. graminis f. sp. tritici populations from different Volga region areas at the seedling stage. Specific DNA primers were used to identify resistance genes (Sr2, Sr24, Sr25, Sr26, Sr28, Sr31, Sr32, Sr36, Sr38, Sr39, and Sr57). Highly resistant cultivars (30 from 126) were identified. In bread wheat cultivars, the genes Sr31 (in 19 cultivars), Sr24 (in one cultivar), Sr25 (in 15 spring wheat cultivars), Sr28 (in six cultivars), Sr38 (in two cultivars), and Sr57 (in 15 cultivars) and their combinations—Sr31 + Sr25, Sr31 + Sr38, Sr31 + Sr28, Sr31 + Sr57, Sr31 + Sr28 + Sr57, and Sr31 + Sr24—were identified. The obtained results may be used to develop strategies for breeding rust-resistant cultivars.