Increasing zinc (Zn) and iron (Fe) levels in edible parts of plants, known as biofortification, is seen a sustainable approach to alleviate micronutrient deficiency in humans. Wheat is recognized as a prioritized choice for biofortification. This study performed high-resolution genome wide association studies (GWAS) for grain zinc (GZnC) and iron concentrations (GFeC) by three different models using 166 wheat cultivars and 373,106 polymorphic markers from wheat 660K and 90K SNP arrays. Totally, 25 and 16 stable loci were detected for GZnC and GFeC, respectively. Twenty eight promising candidate genes were identified for Zn/Fe uptake (8), transport (11), storage (3) and regulations (6) based on gene annotations, homology analysis, and expression data. Of them, 11 genes were putative wheat orthologs of known Arabidopsis and rice genes related to Zn/Fe homeostasis. These genes together with others identified by previous meta-QTL studies were further screened for natural variations in 641 resequencing wheat accessions. Eventually, 77 gene-specific SNPs corresponding to 36 genes including six known genes as internal control, were selected as target loci for developing a genotyping by target sequencing (GBTS) platform, also named wheat biofortification liquid chip. The chip developed by multiplexing PCR technology has three advantages. 1) High throughput; the 77 target loci can be genotyped by one amplification assay. 2) Low cost; each datapoint is about $0.05, which is significantly lower than the cost by KASP and STARP. 3) High accuracy; the chip results of six reference genes were completely consistent with the known genotypes of these loci in a large panel containing 452 wheat accessions. Candidate-gene association study (CGAS) in this panel was performed to identify 23 and 21 associated SNPs, corresponding to 19 genes for GZnC and 15 for GFeC, respectively. Due to its low cost, flexibility and moderate to high genomic predictability, the current biofortification liquid chip v1.0 can be used immediately in breeding for gene stacking or genomic selection, and will be easily updated to an advanced version in the future. The germplasm, genes and the genotyping platform reported in this study will accelerate the release of biofortified wheat.