Metal, primarily Fe and Zn, deficiencies affect over half of the world's population. Human diets with prevalent cereal products cause micronutrient malnutrition. Biofortification is one of the most effective approaches to alleviate malnutrition. Spring wheat genetically stable (M7) mutant lines developed with 100 and 200 Gy gamma treatments to broaden genetic variation and search for new resources were analyzed for nutritionally important minerals (Ca, Mg, K, Fe, and Zn), their bioavailability, and grain protein content (GPC). The variation was 172.3–883.0 mg/kg for Ca, 472.9–1088 mg/kg for Mg, 3128.6–5487.5 mg/kg for K, 40.9–89.0 mg/kg for Fe, and 22.2–89.6 mg/kg for Zn. In mutant lines, among the investigated minerals, the highest increases in concentrations were observed in Fe, Zn, and Ca when compared to the parent. Some mutant lines, mostly in the 100 Gy-derived germplasm, had two to three times higher Fe, Zn, and Ca concentrations, lower phytic acid concentration (1.4–2.1 times), and 6.5–7% higher GPC compare to the parent. Variation was detected for the Ca:Phy, Mg :Phy, Phy:K, Phy:Fe, and Phy:Zn molar ratios, (1.27–10.41, 5.05–18.68, 1.66–4.87, 1.40–5.32 and 1.78–11.78, respectively). The results showed how the genetic variation could be generated through radiation and be useful to develop biofortification by micronutrient varieties with their appropriate bioavailability to overcome malnutrition.