Spike-layer uniformity (SLU), the consistency of the spike distribution in the vertical space, is an important trait. It directly affects the yield potential and appearance. Revealing the genetic basis of SLU will provide new insights into wheat improvement. To map the SLU-related quantitative trait loci (QTL), 300 recombinant inbred lines (RILs) that were derived from a cross between H461 and Chinese Spring were used in this study. The RILs and parents were tested in fields from two continuous years from two different pilots. Phenotypic analysis showed that H461 was more consistent in the vertical spatial distribution of the spike layer than in Chinese Spring. Based on inclusive composite interval mapping, four QTL were identified for SLU. There were two major QTL on chromosomes 2BL and 2DL and two minor QTL on chromosomes 1BS and 2BL that were identified. The additive effects of , and were all from the parent, H461. The major QTL, and , were detected in each of the conducted trials. Based on the best linear unbiased prediction values, the two loci explained 23.97% and 15.98% of the phenotypic variation, respectively. Compared with previous studies, the two major loci were potentially novel and the two minor loci were overlapped. Based on the kompetitive allele-specific PCR (KASP) marker, the genetic effects for were validated in an additional RIL population. The genetic effects ranged from 26.65% to 32.56%, with an average value of 30.40%. In addition, showed a significant ( 0.01) and positive influence on the spike length, spikelet number, and thousand kernel weight. The identified QTL and the developed KASP marker will be helpful for fine-mapping these loci, finally contributing to wheat breeding programs in a marker-assisted selection way.