In order to explore genetic loci that associated with grain number per spikelet in bread wheat (Triticum aestivum L.), a genome-wide association study was conducted using the wheat 55K genotyping assay in a diverse panel of 384 wheat genotypes. Field trials for grain number per spikelet were conducted in one location for three consecutive years. A total of 142 significant SNPs were detected for grain number per spikelet, each of which contributed the phenotypic variation from 3.27% to 6.09%. Of these, eight SNPs were detected in two or three environments. Three SNPs (AX-109986855, AX-109875224 and AX-109843323) were located on the 523.12～526.25 Mb on chromosome 2D, and the physical distance between two SNPs AX-111054388 and AX-110671159) was only 0.62 Mb on chromosome 2B. Significant differences for grain number per spikelet grouped by superior and inferior alleles in an SNP were detected (P < 0.01) across the three environments in each of these eight SNPs. For example, cultivars carrying the G/G allele at AX-109843323 on chromosome 2D resulted in an increase of 0.32, 0.37 and 0.39 grains per spikelet than that of cultivars with the C/C allele in three environments, respectively. The percentages of cultivars carrying each of the superior alleles at the eight SNPs were varied from 5.20% to 76.8%. Seven superior alleles were present with a percentage of less than 45.00% in the accessions. Furthermore, the grain number per spike in these cultivars with superior alleles (ranging from 48.45 to 53.61) among the eight SNPs were higher than that of cultivars without superior alleles (ranging from 45.04 to 47.37). Significant association in the number of favorable alleles to grain number per spikelet and grain number per spike were observed (r = 0.97 and 0.94, respectively; P < 2.0E-4). These loci associated with grain number per spikelet are useful for improving grain numbers per spike in wheat.