Technical Abstract: RNA interference (RNAi) is a promising antiviral methodology. We recently demonstrated that retroviral vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) can be effective in reducing replication of other retroviruses in chicken cells. In this study, similar RNAi vectors are shown to inhibit replication of the avian herpesvirus, Marek’s disease virus (MDV), and its close relative, herpesvirus of turkeys (HVT). Cells expressing shRNA-mirs targeting the MDV or HVT gB glycoprotein gene or the ICP4 transcriptional regulator gene show significant inhibition of viral replication. Not only are viral titers reduced, but observed plaque sizes are significantly smaller when the virus is grown on cells in which RNAi is effective. We also describe a modified retroviral delivery vector that expresses a shRNA-mir containing up to three RNAi target sequences and present results using this vector with multiple targets within the MDV gB gene or within both the gB and ICP4 genes. This multi-target vector may be valuable for eventual in vivo applications in reducing possible escape mutations in the target virus.