SummarySoybeans (Glycine max) are a preferred source of plant‐based proteins and oil in various applications in foods and feeds. When soybean is converted to soybean meal (SBM), the quality is determined by the protein content and its digestibility. Protein digestibility is reduced by anti‐nutritional factors like trypsin inhibitors (TI) and processing. The abundant genetic variations in soybean germplasm affect the quantitative trait of protein content. This study analysed the effect of location and planting dates on genetically identical soybeans grown in North Carolina (NC) and then compared the nutritional traits to similar commonly grown soybean varieties in other locations in the USA. The soybeans were analysed using NIRS and wet chemistry. It was found that soybean protein content ranged from 38.14% to 44.92% among the different varieties grown in three different locations in NC. Similarly, TI levels ranged between 28.22 and 33.11 mg g−1 among these varieties. The same varieties, when planted as either a full season or double crop, had different TI levels and were also impacted by planting location. However, there were no significant differences in the TI levels between the varieties and planting seasons. Furthermore, no significant difference in protein content among the varieties within the planting dates was found. Compared to soybeans grown in other parts of the USA, the average crude protein (CP) content was similar to those grown in Arkansas and Minnesota. The TI levels were significantly higher than those grown in Ohio, similar to that of Arkansas, and significantly lower than that of Minnesota.