Studying the long-term effect of elevated atmospheric CO₂ concentration (e[CO₂]) on wheat (Triticum aestivum L.) over multiple generations has received increasing attention. Here, five wheat cultivars were grown under ambient CO₂ concentration (a[CO₂], 400 ppm) and e[CO₂] (800 ppm), respectively, for three consecutive generations (G1 to G3) under two nitrogen (N) levels (1N and 2N). Compared to plants grown under a[CO₂], e[CO₂] increased shoot biomass and grain yield (GY) over three generations and the enhancement was greater in G3 than in G1. However, plant N concentration was lowered by e[CO₂] and the reduction was not mitigated by higher N supply. The carbon (C) concentration significantly increased in leaf and stem but decreased in grain, indicating an inhibited C translocation to grain under e[CO₂]. Most importantly, these negative impacts were exacerbated in G3. Concentrations of mineral nutrients in grain were significantly lowered by e[CO₂] with larger reduction in G3 than in G1 in some micronutrients such as Zn, Cu and Fe. These findings suggest that long-term exposure to e[CO₂] sustained the positive effects on plant growth and production but aggravated the reduction of grain quality over multiple generations. Among the five cultivars, 325Jimai showed the greatest increase in shoot biomass and GY, and a greater sink capacity compared with the other cultivars, indicating its potential for future breeding strategies.