The grain hardness index (HI) is one of the important reference bases for wheat quality and commodity properties; therefore, it is essential and useful to identify loci associated with the HI in wheat breeding. The grain hardness index of the natural population including 150 common wheat genotypes was measured in this study. The phenotypic data diversity of HI based on four environments and the best linear unbiased prediction (BLUP) was analyzed. The results showed that the grain HI of the natural population ranged from 15.00 to 83.00, the variation range was from 5.10% to 24.44%, and the correlation coefficient was 0.872–0.980. BLUP value was used to grade and assign the grain HI to hard wheat, mixed wheat, and soft wheat, and the assigned phenotypes were used for genome-wide association analysis. Two types of grain hardness index phenotypic values were used for genome-wide association analysis (GWAS) using a 55K SNP array. A total of five significant association loci (p < 0.001) were excavated, among which four loci could be detected in three or more environments. They were distributed on chromosomes 1A and 7D, and the phenotypic contribution rate was 7.52% to 10.66%. A total of 48 sites related to grain hardness were detected by the assignment method, among which five were stable genetic sites, distributed on chromosomes 1A(2), 3B(1), 4B(1), and 7D(1), with phenotypic contribution rates ranging from 7.63% to 11.12%. Of the five loci detected by the assignment method, two stable loci were co-located in the phenotypic mapping results of the hardness index. One of the loci was consistent with previous reports and located on chromosome 1A, and one locus was unreported on chromosome 7D. Therefore, it may be a feasible attempt to use the assignment method to conduct genome-wide association analysis of the grain hardness index. In this study, a total of five genetic loci for grain hardness stability were excavated, and two of the loci were located in the two phenotypic values, two of which were not reported.