To analyze the genetic variability and select elite germplasm of isoflavone content in soybean seed, 213 accessions, including 181 cultivated and 32 wild soybean originating from HuangHuaiHai ecological region, were tested by high performance liquid chromatography (HPLC) technique. The results showed that the linear range of isoflavone components were all between 2.5-80.0 μg/mL, and the applied method possessed good linearity, precision and accuracy. A significant difference was found in isoflavone concentration among the different soybean accessions. Identification of 213 soybeans showed that a large genetic variation in total isoflavone and its components existed in both cultivated and wild soybeans. The range of total isoflavone in cultivated soybean was 1462.6-6115.5 μg/g, with an average of 3558.2 μg/g, while the total isoflavone in wild soybean was 3896.1-7440.4 μg/g, with an average of 5182.4 μg/g. The average content of total isoflavone in wild soybean seed is much higher than the cultivated seed. From the accessions, four elite germplasm with high isoflavone content (>6000 μg/g) were screened out (one elite accession content was above 7000 μg/g), and could be used to increase isoflavone content in soybean breeding.