Panicle length is one of the important factors influencing rice yield. In rice breeding, study of quantitative trait loci (QTL) of panicle length and their epistasis effects in rice is important. In this study, we used 16 secondary single segment substitution lines and 15 double segment pyramiding lines with a Huajingxian 74 background as mapping populations to study QTL for panicle length and their gene interactions. We detected 6 QTLs for panicle length, of which 2 (Pl-2 and Pl-10) were previously unreported. Furthermore, after interactions, some loci pairs had different effect strength and opposite direction of each locus, which indicated that the gene pyramiding may result in various interaction patterns. These results are important for improving rice panicle length with molecular marker-assisted breeding.