Tiller number (TN) and spike number per plant (SN) are key components of grain yield and/or biomass in wheat. In this study, an introgression line 05210, developed by introgression of chromosomal segments from a synthetic exotic wheat Am3 into an elite cultivar Laizhou953, showed a significantly increased TN and SN, but shorter spike length (SL) and fewer grain number per spike (GNS) than Laizhou953. To investigate the quantitative trait locus (QTL) responsible for these variations, the introgressed segments in 05210 were screened by SSR markers and one follow-up segregation population was developed from the cross 05210/Laizhou953. The population showed 3:1 segregation ratios for SN, SL and GNS, indicating that QTLs for these traits have been dissected into single Mendelian factors. Bulked segregation analysis showed that the markers located on the 4B introgressed segment were polymorphic between the two bulks. Therefore, they were further analyzed in the F(2) population to construct a linkage map. Three new QTLs, QSn.sdau-4B, QSl.sdau-4B and QGns.sdau-4B, were detected for SN, SL and GNS, respectively, which explained a large portion of the phenotypic variation (30.1-67.6%) for these traits with overlapping peaks. Correlation analysis and multiple-trait, multiple-interval mapping (MMIM) suggested pleiotropic effects of the QTL on SN, SL and GNS. Therefore, the QTL was designated as QSn.sdau-4B. By a progeny test based on F(3) families using SN, the QTL was mapped as a Mendelian factor to the proximal region of 4BL. It is a key QTL responsible for variation in spike number and size, which had not been reported previously. Thus, it is an important QTL for wheat to achieve high and stable biomass and grain yield. Dissection and mapping of this QTL as a Mendelian factor laid a solid foundation for map-based cloning of grain yield-related QTLs in wheat.