Atmospheric carbon dioxide concentrations ([CO₂]) are increasing, but little is known about how this will affect macronutrient (nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg)) accumulation and partitioning in the aboveground biomass (AGB) for different hard spring wheat genotypes. We examined the responses of six spring wheat genotypes (‘Discovery’, ‘Duchess’, ‘Reliance’, PFR-3026, PFR-3019, PFR-2021) to two CO₂ levels (ambient [aCO₂] and elevated [eCO₂]) and six nitrogen rates (N; 1–10 mM), at the stem elongation growth stage of wheat grown in controlled environment chambers. The AGB yield increased by 35.2% with increasing [CO₂] when N rate was >2 mM. Increasing N supply also increased AGB by up to 3.2-fold over the entire N range applied. The AGB responses to N differed among the genotypes, being lowest for PFR-3019 (7.71 ± 0.11 g/pot) and highest for PFR-2021, PFR-3026 and Duchess at 8.84 ± 0.11 g/pot at both CO₂ levels. Macronutrient concentrations decreased with eCO₂ by 28.0% for Ca to 17.4% for P and K. Nevertheless, absolute nutrient uptake was higher for eCO₂ treatments, because the AGB increase (20.0–52.0%) was proportionally higher than the 4.0–28.0% increase in nutrient uptake. The AGB non-response to [CO₂] at N rates <2mM indicates that this nutrient deficiency was more limiting than the effects of CO₂ level. Therefore, the impact of eCO₂ in the future will depend on N fertilizer management. These results suggest that critical nutrient concentrations used to diagnose the nutrient status of wheat crops will need to be reassessed for eCO₂ conditions.