Total spikelet number per panicle (TSN) is thought to be one of the most important agronomic traits associated with grain production in rice (Oryza sativa L.). We previously reported the development of 334 introgression lines (ILs) with variations in agronomic traits in the genetic background of indica rice variety IR64. Among these, an IL derived from high-yielding rice variety Hoshiaoba showed significantly higher TSN than IR64. We therefore have conducted a quantitative trait locus (QTL) analysis for TSN in an F2 population of IL × IR64 to ascertain the genetic basis for the high TSN of the IL. The analysis revealed the presence of a QTL for TSN on chromosome 7, where several QTLs for grain number have been previously reported. We developed a near-isogenic line (NIL) for this QTL by using DNA marker-assisted selection and characterized its effect. The NIL showed significantly higher TSN than IR64. These results suggest that the QTL and the NIL will be useful materials in breeding programs aimed at increasing TSN.