Abstract: In order to determine the difference in nodulation ability of soybean varieties（lines） bred in the Huang-Huai-Hai region, 143 soybean varieties（lines） were used as materials grown in the Huang-Huai-Hai region during 1970-2020, and inoculated with rhizobia strain USDA110 under pot conditions. Root nodule dry weight of a single plant was used as the identification index of nodulation ability to screen soybean germplasms with high or low nodulation ability. Meanwhile, the nodulation differences of the tested materials from different provinces and cities in the Huang-Huai-Hai region were compared. The correlation between the nodulation differences and yield, released year, protein content, oil content and total protein-oil content were analyzed. The results showed that the maximum number of nodules per plant among different soybean varieties（lines） tested was 35, the minimum was 6, and the average was 17. The maximum dry weight of root nodules per plant was 124.89 mg, and the minimum was 26.44 mg, with an average value of 53.81 mg. The top 5% varieties（lines） with the highest nodulation ability were selected as the parents for cultivating varieties with high nodulation ability, including Andou 1311, Guanyundasili, Yandali, Taifeng 6, Shangdou 7, Huangdou ZDD08405 and 08Yguan 205. The dry weight and number of root nodules per plant in Jiangsu province were the largest, 64.30 mg and 20, respectively. The dry weight of root nodules in Shandong province was the smallest, 48.50 mg. The number of root nodules in Shanxi and Anhui provinces was the smallest, the minimum number was 15, and the dry weight of nodules per plant was positively correlated with the seed protein content. The research results can provide useful nitrogen-fixing parent materials for soybean breeding in Huang-Huai-Hai region.