Epistasis plays an important role in the genetic basis of rice yield traits. Taking interactions into account in breeding programs will help the development of high-yielding rice varieties. In this study, three sets of near isogenic lines (NILs) targeting three QTLs for spikelets per panicle (SPP), namely qSPP1, qSPP2 and qSPP7, which share the same Zhenshan 97 genetic background, were used to produce an F 2 population in which the three QTLs segregated simultaneously. The genotypes of the individual F 2 plants at the three QTLs were replaced with three markers that are closely linked to the corresponding QTLs. These QTLs were validated in the F 2 and F 3 popula- tions at the single marker level. qSPP7 exhibited major pleiotropic effects on SPP, plant height and heading date. Multifactor analysis of variance was performed for the F 2 population and its progeny. Additive × additive interaction between qSPP2 and qSPP7 had significant effects on SPP in both the F 2 population and its progeny. Both additive and additive × additive interactions could explain about 73% of the total SPP phenotypic variance. The SPP performance of 27 three-locus combinations was ranked and favorable combinations were rec- ommended for rice breeding in different ecosystems. Taking interactions into account in breeding programs will help the development of high-yielding rice varieties. In this study, three sets of near isogenic lines (NILs) targeting three QTLs for spikelets per panicle (SPP), namely qSPP1, qSPP2 and qSPP7, which share the same Zhenshan 97 genetic background, were used to produce an F2 population in which the three QTLs segregated simultaneously. The genotypes of the individual F2 plants at the three QTLs were replaced with three markers that are closely linked to the corresponding QTLs. These QTLs were validated in the F2 and F3 popula tions at the single marker level. qSPP7 showed major pleiotropic effects on SPP, plant height and heading date. Multifactor analysis of variance was performed for the F2 population and its progeny. Additive x additive interaction between qSPP2 and qSPP7 had significant effects on SPP in both the F2 population and its pro Both SP and SPP phenotypic variance. The SPP performance of 27 three-locus combinations was ranked and favorable combinations were rec- ommended for rice breeding in different ecosystems.