Kernel weight is one of the three elements contributing to the wheat yield, and kernel size is highly correlated with kernel weight. Therefore, the study of kernel size has a great significance to improve the wheat yield. The TaGS2 gene positively regulates kernel size and kernel weight in wheat. In order to further explore allelic variation of TaGS2 gene in wheat, specific primers were designed to amplify the genomic DNA sequence of three TaGS2 homoeologues and identify the polymorphisms in 12 samples of 268 wheat cultivars from the Yellow and Huai wheat regions. A deletion of three nucleotides (CTT) at position 3,002 bp in the fourth exon of TaGS2-2B genomic DNA sequence was detected in some cultivars, resulting in a deletion of one phenylalanine (F) at the 306 position. Both alleles without and with the 3 bp deletion was designated as TaGS2-2B-a and TaGS2-2B-b, respectively. In wheat cultivars carrying TaGS2-2B-a allele, thousand-kernel weight, kernel length, kernel width, kernel area, and kernel circumference were significantly higher than those of wheat cultivars harboring TaGS2-2B-b allele. Collectively, this study provided a relatively superior allele (TaGS2-2B-a) with positive effects in increasing kernel weight in wheat cultivars.