To create genetic materials with breeding value and to identify candidate regions affecting the grain number per spike in wheat, this study used near-isogenic lines (NILs) constructed from hybrids between the octoploid Thinopyrum ponticum and common wheat cultivars Hengguan 35 and Kenong 199 as research materials. Six yield-related traits, including plant height, effective tiller number, spike length, spikelet number per spike, grain number per spike, and grain yield per plant, were phenotypically evaluated. The 660K SNP array was used for whole-genome scanning of near-isogenic lines (NILs) with phenotypic differences to analyze polymorphic SNPs loci and consistent physical intervals between two pairs of NILs. Important candidate genes affecting the grain number per spike were predicted based on gene function annotation and gene expression analysis within the candidate regions. The