Transgenic Nicotiana benthamiana plants harboring coat protein (CP) gene of Zucchini green mottle mosaic virus (ZGMMV) were generated for virus-resistant screening and complementation analysis of related viruses for environmental safety assessment (SA) of living modified organism (LMO) purposes. Transformation of leaf disc of N. benthamiana was performed by using Agrobacterium-mediated method and the pZGCPPGA748 containing the ZGMMV CP and NPTII genes. Two kinds of transgenic homozygous groups, virus-resistant and virus-susceptible N. benthamiana lines, were obtained by screening of challenging homologous virus for T1 generations. These two pathologically different lines can be useful for host-virus interactions and LMO environmental SA.