Calcium is an essential mineral element for the human body. As a major global food crop, wheat's grain calcium content directly affects human nutritional health. Improving the calcium content of wheat grains through biological reinforcement is the most economical and effective way to solve the problem of calcium deficiency in the human body. However, the molecular regulatory mechanism of grain calcium content in wheat is still unclear. This study measured the grain calcium content of 349 wheat accessions and conducted genome-wide association study (GWAS) using a Wheat 660K SNP array. The results showed that a total of 523 significant SNPs were detected, mainly distributed on chromosomes 4A, and 6B, of which 76 SNPs could be detected in 4 or more replicates. Haplotype analysis suggests that GCaC_Hap_4A, and GCaC_Hap_6B may be important gene loci regulating grain calcium content. Using bioinformatics analysis, it is speculated that the TraesCS4A03G0985800 gene may be a candidate gene at the GCaC_Hap_4A locus. This study can provide important reference information for understanding the genetic mechanism of grain calcium content and molecular breeding of wheat.