A strategy for the screening of candidate virus-derived sequences to provide RNA-mediated citrus tristeza virus (CTV) resistance and early selection of virus-resistant citrus is presented. The system is based on the polyethylene glycol- (PEG) mediated cotransformation of protoplasts using virus-derived sequences and green fluorescent protein as a single selectable marker, followed by an in vitro assay of virus inoculation into transgenic protoplasts to determine the level of citrus tristeza virus replication. A cotransformation rate higher than 20% allowed selection of several clones carrying the desired transgenes. Efficient in vitro inoculation of virus in transgenic protoplasts was performed. Tobacco mosaic virus virions were used as a control in order to check citrus protoplast viability. Different CTV replication levels were detected in transgenic clones. Only one clone showed no replication of CTV. Considerations regarding selection of candidate virus-derived sequences and virus challenge of transgenic cells are presented.