Peduncle of wheat is the upmost internode, and its length determines plant height and facilitates spike protrusion, both of which are associated with grain yield. In the previous study, one major QTL QPl.nau-7A controlling wheat peduncle length was identified through QTL mapping with a RIL population derived from the cross "Nanda2419 × Wangshuibai". To validate this QTL, NILs were developed in the backgrounds of Yangmai15, Yangmai17, and Chuanmai42 through marker-assisted selection. In the NIL-derived F2 population, QPl.nau-7A acted as a recessive Mendel factor. Compared with the recurrent parents, all NILs showed significant decreases in peduncle length. In addition, this QTL significantly enhanced grain weight and yield while having no negative impact on spike number per plant, spike length, and grains per spike. These findings showed that QPl.nau-7A is valuable for wheat breeding.