Objective To determine the maintenance and loss of Epstein-Barr virus (EBV) genome during the clonal expansion of the EBV-infected epithelial cells. Methods The epithelial tumor cell line, 293-EBV, in which the EBV genome was observed with green fluorescent protein (GFP) readout. After a dozen of passages, it contained cells with strong or weak GFP expression, and some with complete loss of EBV genome. The cell growth was then continuously observed under a confocal microscope. The cell dividing and GFP expression were also observed during the clonal expansion by being made into very low density. Results The cells moved around due to adherence and mobility, while the GFP expression remained unchanged in the undivided cells. The cells could form compact or loosen clones. The EBV genome easily persisted in those clones when cells were growing compactly. As the cell number increased, the GFP expression became weak or even died away at the sites of low density in the loosen clones. Conclusion EBV-positive epithelial cells are able to sustain the EBV genome during its clonal expansion. The cells maintain EBV genomes by passing them to the daughter cells after replication. When the cells unsuccessfully inherit the EBV genome, the daughter cells may lose them which is related to the low cell density as well as the epithelial environment.