Soybean grains are rich in amino acids and are important components of soybean quality, with high nutritional value and physiological functions. Therefore, breeding soybean varieties with high levels of free amino acids can assist in high-quality soybean breeding and is of significant practical importance for innovative development of the soybean industry. In this study, 264 soybean varieties were analyzed using high-performance liquid chromatography (HPLC) to determine the levels of four free amino acids: arginine, glycine, glutamic acid, and lysine in dried soybean grains. The results showed that arginine had the highest content among the free amino acids, followed by glutamic acid, and glycine had the lowest content. Three soybean varieties with high levels of these four free amino acids were selected: Haimen Yang 104, Liaoxian Bean 12, and Guanyun Dasiyu. Whole-genome association analysis was performed combining the phenotype and genotype data of the natural soybean population for the four amino acids, and significant SNP loci associated with the four amino acids in soybean were identified. This provides a basis for subsequent identification of candidate genes related to high levels of amino acids in soybeans, thereby accelerating soybean variety improvement and facilitating molecular breeding of soybeans.