<p>Global climate changes, including elevated carbon dioxide (eCO₂) and heat stress (HS), have been reported to reduce crop yields and deplete beneficial nutrients in grains. In this study, we investigated the effects of eCO₂ (650 ± 30&#xa0;ppm) and varying nitrogen treatments on stress tolerance, nitrate assimilation, and grain quality in contrasting wheat cultivars: HD2967, HD2329, HI1500, and GW322, under HS conditions (38 ± 3&#xa0;°C). Wheat plants grown under nitrogen-sufficient conditions (G₂) and eCO₂ exhibited enhanced chlorophyll content, longer flag leaves and spikes, and improved photosynthetic and transpiration rates. eCO₂ was found to mitigate the adverse effects of HS, with the positive effects being more pronounced in thermotolerant cultivars HD2967 and HI1500 than in thermosusceptible cultivars HD2329 and GW322. A more developed root system architecture (RSA) was observed in plants grown under G₂ in response to eCO₂. Excess nitrogen also modulated the activities of nitrate assimilatory enzymes, including nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (NADH-GOGAT), under combined eCO₂ and HS conditions. A similar pattern was seen in antioxidant enzymes linked to thermotolerance, such as superoxide dismutase (SOD) and catalase (CAT). However, grain quality—measured in terms of starch, lipid, protein content, and the activity of the starch-degrading enzyme α-amylase—was compromised under eCO₂ and HS in plants grown under nitrogen-deficient conditions (G₁). Thermosusceptible cultivars were more severely affected compared to thermotolerant ones. The findings from this study will help in identifying desirable donor lines through screening of germplasm and will contribute to future wheat breeding programs aimed at developing climate-resilient crops.</p>