Evaluation of genetic biodiversity for micronutrients is crucial for breeding high-quality crops and addressing the negative impacts of mineral deficiencies. The objectives of this research were to assess genetic variation and the relationship between grain Fe and Zn levels and agronomic traits in a diverse collection of wheat varieties. Additionally, the study aimed to determine the correlation between microsatellite markers (SSR) and micronutrient quantities. A total of 42 genotypes (Iranian commercial cultivars, landraces, and Afghan and Swiss varieties) were evaluated over a two-year period. Fe and Zn levels were measured using two semi quantitative staining assays and atomic absorption spectrophotometry (AAS) facility. Semi-quantitative staining methods and AAS showed high correlations for micronutrient contents. Landraces exhibited higher Fe (63.79 mg/kg) and Zn (44.76 mg/kg) but lower grain yield compared with commercial cultivars. Heritability estimates ranged 53%-79.43%, suggesting that genetic variance played a higher contribution in the phenotypic variation of traits than environmental factors. Notably, Fe content displayed significant correlations with days to maturity. Canonical correlation analysis (CCA) revealed that Zn content was correlated with four agronomic traits. Evaluation of genetic diversity using SSR markers demonstrated high genetic variation among the genotypes tested. The analysis of polymorphism information content (PIC) indicated that SSR primers had an average PIC of 0.75, with the Xgwm192 primer exhibiting higher PIC than others. Several SSR markers revealed association with micronutrient content that can be used in marker-assisted selection (MAS) programs aimed at selection of high micronutrient genotypes. In conclusion, the findings underscored the substantial genetic diversity present in micronutrient levels among global wheat genotypes, the potential of landraces for micronutrients biofortification of wheat cultivars through cross hybridization, the utility of staining methods for screening high/low micronutrient genotypes, and use of microsatellite markers for marker-assisted breeding aiming to micronutrient improvement in breeding programs.