Totally, 48 loci responsible for six spike-related traits were identified in wheat, and a major locus QGl-4A for grain length was mapped and validated for marker-assisted selection in breeding. Wheat yield is determined by the number of spikes, number of grains per spike (GN), and one-thousand kernel weight (TKW), among which GN and TKW are greatly related to the spike development and thus the spike-related traits, including spike length (SL), number of spikelet per spike (SN), grain length (GL) and grain width (GW). To identify the key loci governing the spike-related traits (SL, SN, GN, TKW, GL and GW), we conducted the quantitative trait loci (QTL) analysis combined with wheat 660K SNP chip and Kompetitive allele-specific PCR (KASP) assay, using the F2 and F2:3 populations derived from Luohan6 (LH6) with big spike and grain and Zhengmai366 with small spike and grain, and identified a total of 48 QTLs on 18 chromosomes. Moreover, a major stable QTL for GL on chromosome 4A, designated as QGl-4A, was mapped into a 0.37 cM interval between KASP markers Xib4A-10 and Xib4A-12, corresponding to 20 Mb physical region in the Chinese Spring genome. This QTL explained 17.30% and 5.12% of the phenotypic variation for GL in the F2 and F2:3 populations. Further association analysis of flanking markers Xib4A-10 and Xib4A-12 in 192 wheat varieties showed that these two markers could be used for marker-assisted selection in breeding. These results provide valuable information for map-based cloning of the target genes involved in the regulation of spike-related traits in common wheat.