Simian varicella virus (SVV), the primate counterpart of varicella zoster virus, causes varicella (chickenpox), establishes latency in ganglia and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing the enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and infected monkeys. Herein, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo One harbors eGFP fused to the N-terminus of open reading frame (ORF) 9 (rSVV.eGFP-2a-ORF9) and another in which eGFP was fused to the C-terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike the SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (IT) or IT plus intravenous (IV) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) at 10 days post-infection (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis.IMPORTANCE SVV infection in non-human primates has served as an extremely useful animal model to study VZV pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus.