Molecular mechanisms of Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named "Rainbow-KSHV," that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication.IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented.