Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating fungal disease that affects wheat globally. The planting of resistant cultivars is the most cost-effective strategy for controlling this disease. The Huanghuai region, as a major wheat-growing area, plays a crucial role in the spread and prevalence of wheat stem rust in China. In this study, 64 wheat accessions from this region were tested at the adult stage against two major Pgt races, 34MKGQM and 21C3CTHQM. DNA markers associated with the known resistance genes Sr31, Sr24, Sr25, Sr26, and Sr38 were measured to determine their presence in the tested accessions. In the 2023 field tests, 5 (7.8%) accessions were immune to 21C3CTHQM and 34MKGQM, while 35 (54.7%) and 39 (60.9%) were moderately resistant and resistant, respectively. The remaining 20 (30.7%) accessions were moderately susceptible and susceptible. In the 2024 tests, 12 (18.8%) and 14 (21.9%) entries were immune to both races; 29 (45.3%) and 30 (46.9%) were moderately resistant and resistant, respectively. Only two cultivars, Xinong 816 and Yimai 211, were immune in both years, and three entries showed some degrees of resistance in both years. Seven cultivars, including Zhongzhimai 23, Longxing 1, Yunong 937, Huaguan 301, Wanke 800, Shaanhe 285, and Yunong 612, showed increased susceptibility. DNA markers showed that 30 entries carried Sr31, while 6 entries carried Sr38. Genes Sr24, Sr25, and Sr26, which confer good resistance to the globally prevalent cultivars TKTTF and TTTRF, were absent from the set of tested entries. While this study surveyed the resistance levels of a cross-section of wheat from the southern part of the Huanghuai region and confirmed the presence of two known resistance genes, the basis of immunity or high levels of resistance in several lines remains obscure.