Grain weight (GW) is one of the most important targets for grain yield in rice breeding. In previous studies, two quantitative trait loci (QTLs) for GW, gw8.1 and gw9.1, have been identified using progeny derived from a cross between the japonica cultivar Hwaseong and Oryza rufipogon (IRGC 105491). To test whether these quantitative trait loci (QTLs) have an epistatic interaction, we developed an F₂ population by crossing two nearly isogenic lines (NILs) harboring gw8.1 and gw9.1. Simple sequence repeat (SSR) markers tightly linked to the QTLs were used to select F₃ QTL-NILs from the F₂ population. A two-way ANOVA revealed an epistatic interaction between the two QTLs in the F₂ population (P = 0.0084). This interaction was confirmed by an analysis of F₃ QTL-NILs indicating that both QTLs are involved in the same genetic mechanism controlling GW. The gw8.1 QTL was further mapped between two SSR markers, RM23204 and RM23211, which are 110.1 kb apart. To our knowledge, this is the first report using QTL-NILs to reveal an epistatic interaction between QTLs for GW.