QTL mapping based on the primary mapping population could precisely characterize and locate genes underlying specific agronomic traits, which was also true for both major and moderate/minor QTLs55. Previous studies have confirmed that tagged or cloned genes were near their original QTL positions (logarithm of the odds (LOD) peak)56–67. In this study, qKnps-4A was repeatedly identified using MapQTL 6.0, IciMapping 4.1, and QTLNetwork 2.0, based on mathematical models of composite interval mapping (CIM), inclusive composite interval mapping (ICIM) and the mixed linear model approach (MLMA), respectively. In addition, the KNPS values of the 188 KJ-RILs and their parental lines were evaluated in 10 different environments. Based on the 10 environmental phenotypic values along with our high-density genetic map, we confirmed QTL peak position with the aforementioned QTL mapping software and found the Ax-109844107–Ax-110540586 overlapping confidence interval, which spanned 3.23 Mb (4 A:680 398 739–4 A:683 638 403) in physical position (Table 1; Fig. 4; Figures S5, S6, S7). In addition, qKnps-4A has been mapped to 4 A:664 209 916–4 A:736 771 450 in both the WY and WJ populations4 and to 4 A:632 864 778–4 A:688 093 018 in the Zhou 8425B/Chinese Spring population54 (Supplementary Table 7). These coincidences across different genetic backgrounds, multiple environments and diverse QTL models strongly supported the hypothesis that the genes underlying qKnps-4A are likely located within 4 A:680398739–4 A:683638403. One of the 65 predicated genes within this interval might be the candidate gene for qKnps-4A (Figure S8). This information is very valuable for future high-resolution mapping and map-based cloning of qKnps-4A.