AbstractAs an important component of wheat (Triticum aestivum L.) yield, 1000‐kernel weight (TKW) has played a crucial role in yield improvement in recent decades. Marker‐assisted selection is an effective tool for improving quantitative traits; however, most markers have not been effectively applied in TKW improvement despite the identification of many loci associated with TKW. In this study, association mapping was performed using local cultivated varieties, and a previous linkage analysis was integrated to identify 10 candidate intervals. The corresponding kompetitive allele‐specific PCR markers associated with TKW were successfully developed and screened in 1153 F4 breeding lines. Three markers, namely, Ktkw‐5B2, Ktkw‐5D, and Ktkw‐7D, were proved to be significantly related to TKW according to large‐scale association mapping, and their ability to increase TKW was enhanced in combination. We applied the three markers in 150 F6 breeding lines and obtained 25 high‐TKW lines, indicating the availability in marker‐assisted selection. The TKW‐increasing allele for Ktkw‐5D was heavily selected in the present breeding program, while that for Ktkw‐5B2 was low and for Ktkw‐7D tended to increase after 2000. Furthermore, strategies for gene exploration in the three significant intervals were proposed. These results are expected to provide an efficient route for improving TKW in wheat high‐yield breeding.