Nutrient and trypsin inhibitor (TI) content of soybeans can be influenced by genetics, planting site, and maturity group, among other factors. Heat treatments during soybean processing reduce most of TI, but the initial content of TI and other thermoresistant antinutritional factors affect soybean meal quality for use in poultry feed. In this study, we assessed the CP, Lys, Met, Thr, and Trp content (%) as well as TI (mg/g) in four commonly grown varieties of soybeans (AG56X8, AG69X0, S53-F7X, and S62XT09, labeled here A to D, respectively) planted in 3 locations of North Carolina (counties Robeson, Beaufort, and Rowan, labeled as X, Y, Z, respectively) in two maturity groups double crop (growing two successive crops in one year) and full season (growing one traditional crop in a year). The 4 x 3 x 2 factorial arrangement of treatments resulted in 24 treatment combinations. A completely randomized design with four plot replicates was used. Raw soybean samples were cleaned from foreign material and standardized to the USDA soybean grading system to be grade 3 or 2. These samples were either submitted to wet chemistry by AOAC methods or ground in a Retsch grinder at 16000 rpm with a sieve opening of 0.5 mm to be analyzed by NIRS. NIRS spectra were collected in a FOSS DS 2500, and data were read with the Soya package AMINONIR® RED (Evonik). Data were analyzed using a three-way ANOVA while mean separation was done by Tukey’s or student’s t-test. All data were standardized to dry matter content before statistical analysis. Results by wet chemistry and NIRS gave similar results (P<0.001) with a strong correlation between both methods (r = 0.99, 0.97, 0.71, 0.97, 0.88, and 0.90 for CP, Lys, Met, Thr, Trp, and TI, respectively). Results of both datasets indicated similar three-way interaction effects (P<0.01). Soybean varieties B and C in location X had higher CP, Lys, Met, Thr, and Trp content than crops of Y and Z locations, and results did not vary according to maturity group. The highest TI content was observed in variety D (S62XT09) and the lowest in variety A (AG56X8). In general, beans from full season had higher nutrient content and lower TI than those from double-crop. The lowest CP and amino acid content and the highest TI were observed in variety D (S62XT09) in Y and Z locations in both maturity groups. There was (P<0.001) a negative correlation (average r = -0.75) between TI and nutrient content, except for Met (P>0.05).