Zinc (Zn) deficient in the cereal crops, especially for wheat, is one of the main reasons for human Zn malnutrition. Zinc biofortification of high yield wheat cultivars is a key way to meet with the dual demand for grain yield and quality. In this study, a three-year field trial has been conducted to analyze the distribution of Zn and its co-localization with P and S and speciation in grain tissues of wheat cultivars with contrasting grain Zn concentration using synchrotron micro X-ray fluorescence imaging (μ-XRF) and X-ray absorption near edge structure (XANES). Results showed that Zn distribution in the high-Zn cultivar was enhanced in the crease tissue, the aleurone layer, the embryo and the endosperm compared to that of the low-Zn cultivar. High-Zn cultivar had less colocalization of Zn with P and the lower P/Zn ratio, but more Zn colocalized with S than the low-Zn cultivar. XANES results also indicated that the proportion of Zn-phytate was lower in the high-Zn cultivar. Findings of the present study could provide a new perspective for grain Zn biofortification, namely increasing grain Zn bioavailability by selecting the high-Zn wheat cultivar.