Using Ax-110540586 as a probe, the 188 KJ-RILs were divided into two groups, one group with alleles from KN9204 and the other with alleles from J411, to perform mean comparisons regarding KNPS. The positive alleles from qKnps-4A’s increased the KNPS value from 3.0 to 4.6, indicating a tremendous potential for their application in wheat molecular breeding programmes designed to increase yield (Figure S9). To characterize the use of qKnps-4A’s positive alleles in wheat breeding programmes, we dissected the genotypes of 65 KN9204-derived advanced lines/authorized varieties, three parental lines of KN9204, three control varieties from the Winter Wheat Performance Trial of the Northern Huang-Huai Regional Nursery of China, and CS near Ax-109844107–Ax-110540586 (Figure S10). None of KN9204′s three parental lines carried favourable alleles for increasing KNPS, which accounted for the negative alleles of KN9204 at this QTL. Only 3 (4.6%) of the 65 authorized/advanced lines derived from KN9204 lines carried favourable alleles for increasing KNPS. Interestingly, two advanced lines (KN1002-13-10 and O-97) carried heterozygous alleles, which were used to develop the secondary mapping population of Knps-4A via self-cross (data not shown). Of the three control varieties from the Winter Wheat Performance Trial of the Northern Huang-Huai Regional Nursery of China, S4185 carried negative alleles, decreasing KNPS; chromosomal regions of Knps-4A in LX99 and JM22 were recombinant regions with alleles that cannot be categorized as favourable or negative alleles that increase or decrease KNPS; and CS carried favourable alleles, increasing KNPS. These findings indicated that favourable qKnps-4A alleles have not been fully utilized in wheat breeding programmes in the Huang-Huai winter wheat region in China. Wheat breeders should strengthen the selection of qKnps-4A favourable alleles in molecular breeding programmes aimed at the development of high-yield varieties.