Some haplotypes of the sucrose synthase gene TaSus1 are associated with thousand-grain weight (TGW) in wheat (Triticum aestivum L.). However, no mutations have been identified within the gene to test this association. The effects of TaSus1 on grain number per spike (GNS) also are largely unknown. Our previous genome-wide association study identified TaSus-A1 as a candidate gene controlling fertile spikelet number per spike (FSN). In the present study, we generated two independent mutants for the three TaSus1 homoeologs by CRISPR/Cas9-mediated genome editing. The triple mutants displayed lower FSN, GNS, grain number per spikelet (GNST), and TGW than wild-type plants. In 306 hexaploid wheat accessions, two single-nucleotide polymorphisms in TaSus-A1 contributed differently to GNS. Introgression of the two alleles into a wheat genetic background confirmed their effects. The alleles differed in geographical distribution among the accessions.