Stem rust disease caused by the obligate biotrophic fungus Puccinia graminis f. sp. tritici significantly reduces the yield and quality of wheat grain. A total of 92 entries of new Siberian agricultural wheat germplasm were studied with an integrated approach: phenotyping in the field and seedling stage reactions and using molecular markers to identify stem rust resistance genes. Among the tested germplasm, 38% were highly or moderately sensitive to stem rust, and 56% were resistant or moderately resistant. In total, 10 out of 16 molecular markers were positive in 92 entries of the new Siberian germplasm: specific or major genes Sr15, Sr22, Sr23, Sr24, Sr25, Sr31, Sr38 and Sr44 and nonspecific or minor genes Sr2 and Sr57, singly or combined. The Sr31 + Sr44, Sr31 + Sr24 and Sr31 + Sr25 gene pyramids provided effective resistance to natural infection. Since a substantial number of gene pyramids with various degrees of genetic resistance were identified, this wheat germplasm could be useful to pursue a strategy of durable resistance to stem rust in Western Siberia.