The spikelet number per spike (SNS) contributes greatly to grain yield in wheat. Identifying various genes that control wheat SNS is vital for yield improvement. This study used a recombinant inbred line population genotyped by the Wheat55K single-nucleotide polymorphism array to identify two major and stably expressed quantitative trait loci (QTLs) for SNS. One of them (QSns.sau-2SY-2D.1) was reported previously, while the other (QSns.sau-2SY-7A) was newly detected and further analyzed in this study. QSns.sau-2SY-7A had a high LOD value ranging from 4.46 to 16.00 and explained 10.21–40.78% of the phenotypic variances. QSns.sau-2SY-7A was flanked by the markers AX-110518554 and AX-110094527 in a 4.75-cM interval on chromosome arm 7AL. The contributions and interactions of both major QTLs were further analyzed and discussed. The effect of QSns.sau-2SY-7A was successfully validated by developing a tightly linked kompetitive allele specific PCR marker in an F2:3 population and a panel of 101 high-generation breeding wheat lines. Furthermore, several genes including the previously reported WHEAT ORTHOLOG OF APO1 (WAPO1), an ortholog of the rice gene ABERRANT PANICLE ORGANIZATION 1 (APO1) related to SNS, were predicted in the interval of QSns.sau-2SY-7A. In summary, these results revealed the genetic basis of the multi-spikelet genotype of wheat line 20828 and will facilitate subsequent fine mapping and breeding utilization of the major QTLs.