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The grain number per spike (GNPS) is an important yield component, and much attention is given to the increase in GNPS for current yield improvement of common wheat. Here, a panel of 259 pre-breeding lines and elite commercial varieties were collected for the investigation of 12 agronomic traits, especially for spike-related traits, with 2-year replicates. The high correlation between GNPS and kernel number per spikelet (KNS) suggested that the high GNPS trait in our pre-breeding lines was mainly controlled by grain set number per spikelet. Genome-wide association studies (GWAS) using the 660K SNP genotyping assay suggested that a major locus on chromosomes 4BS contributed to the high GNPS trait, which contributed to 33% and 48% of the variation in KNS and GNPS, respectively. A good diagnostic KASP marker AX-109286577 flanking the 4BS locus was developed for easy selection of the large spike trait. Taken together, the results suggested that untapped rare allele variation in our pre-breeding lines can be used for improvement of the yield component of set grain number per spike.