A twin T-DNA system is a convenient strategy for creating selectable marker-free transgenic plants. The standard transformation plasmid, pCAMBIA 1300, was modified into a binary vector consisting of two separate T-DNAs, one of which contained the hygromycin phosphotransferase (hpt) marker gene. Using this binary vector, we constructed two vectors that expressed inverted-repeat (IR) structures targeting the rice stripe virus (RSV) coat protein (CP) gene and the special-disease protein (SP) gene. Transgenic rice lines were obtained via Agrobacterium-mediated transformation. Seven independent clones harbouring both the hpt marker gene and the target genes (RSV CP or SP) were obtained in the primary transformants of pDTRSVCP and pDTRSVSP, respectively. The segregation frequencies of the target gene and the marker gene in the T1 plants were 8.72% for pDTRSVCP and 12.33% for pDTRSVSP. Two of the pDTRSVCP lines and three pDTRSVSP lines harbouring the homozygous target gene, but not the hpt gene, were strongly resistant to RSV. A molecular analysis of the resistant transgenic plants confirmed the stable integration and expression of the target genes. The resistant transgenic plants displayed lower levels of the transgene transcripts and specific small interfering RNAs, suggesting that RNAi induced the viral resistance.