A major and stably expressed QTL for spikelet number per spike identified in a 2-cM interval on chromosome arm 2DS was validated using two populations with different genetic backgrounds. Spikelet number per spike (SNS) plays a key role in wheat yield improvement. Numerous genetic and environmental factors influencing SNS have been documented, but the number of major, stably expressed and validated loci underlying SNS is still limited. In this study, a recombinant inbred line (RIL) population derived from a normal spikelet cultivar and a multiple-spikelet wheat line (with a longer spike with more canonically oriented apical spikelets) was genotyped using a Wheat55K single-nucleotide polymorphism (SNP) array and simple sequence repeat (SSR) markers. SNS was measured for this RIL population in eight environments. Five QTL were each identified in two or more environments. One of them, QSns.sau-2D (LOD = 3.47-38.24, PVE = 10.16-45.68%), was detected in all the eight environments. The QTL was located in a 2-cM interval on chromosome arm 2DS flanked by the markers AX-109836946 and AX-111956072. This QTL, QSns.sau-2D, significantly increased SNS by up to 14.72%. Several genes associated with plant growth and development were identified in the physical interval of QSns.sau-2D. This QTL was further validated by the tightly linked Kompetitive Allele Specific PCR (KASP) marker, KASP-AX-94721936, in two other populations with different genetic backgrounds. The significant correlation between SNS and anthesis date, plant height, spike length, grain number per spike and thousand-grain weight were detected and discussed. These results lay the foundation for fine mapping and cloning gene(s) underlying QSns.sau-2D.