The significant prevalence of zinc insufficiency in people makes it crucial to increase zinc (Zn) content in wheat grains on a worldwide scale. Grain-based diets that were high in zinc help combat the global issue of zinc deficiency. Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted Polymorphism (SCoT) were used to analyze the genetic diversity of 10 Egyptian wheat cultivars in order to find the ones with the highest levels of built-in Zn bio-fortification. The level of polymorphism detected by SCoT was greater than that detected by ISSR. To clarify the influence of Zn foliar treatment on wheat grain production in 2019 and 2020, under conditions of zinc deficiency, Zn foliar application (5 g/L as ZnSO4.7H2O) was administered at two physiological phases (tillering and milking). Foliar treatment greatly raised the zinc content of the granules. A grain production boost of 50% was seen with a Zn content of 55 ppm. Zinc was bio-fortified in the Giza-168, Gemiza-7, and Gemiza-10 cultivars. This means that foliar treatment of ZnSO4 may be a useful and cost-effective method for achieving agricultural bio-fortification.