Producing desirable firmness is important in manufacturing tofu from soybeans. This study’s objective was to explore the environmental impact (location and year) on soybean chemical components and identify the correlations between chemical composition and the firmness of tofu made from soybeans planted in three locations over two years. Seventeen soybean Plant Introductions (PI) from the USDA Soybean Germplasm Collection and eight check varieties were planted in Mississippi, Virginia and Missouri in 2017 and 2018. Protein subunit composition, protein secondary structure, phytic acid content, Ca2+ and Mg2+ content were determined. The result showed that A3 subunit content was strongly correlated with tofu firmness. Environmental factors had a significant influence on some chemical components in soybean seeds as well as tofu texture. The current study confirmed the validity of using A3 peptide as a criterion for estimating tofu firmness in both tofu manufacturing and food-grade soybean trade.