The parasitoid Diachasmimorpha longicaudata (Dl) oviposits into tephritid fruit fly hosts and introduces an entomopoxvirus (DlEPV) that inhibits the host’s immune response and facilitates wasp development. DlEPV is present at very high titers in the adult wasp’s poison gland apparatus (PGA) but the source of this virus population is unknown. Here we test the hypothesis that this population arises by viral replication within the PGA during pupal development; alternatively, this population comes from tissues outside the PGA. Using timed dissections we identified the first appearance and subsequent development of the PGA. We evaluated the appearance of viral DNA by PCR with primers for the DlEPV rifampicin resistance structural gene (rif) and of viral proteins with DlEPV polyclonal antibodies in ELISA to ascertain the onset and progression of viral replication. The PGA first becomes evident 12 h after pupation and develops to its adult-like form by 24 h. The rif primers amplified DlEPV DNA from the PGAs of all pupae tested and the amount of transcript increased dramatically with pupal age. Similarly, an 8-fold increase in viral proteins was observed by ELISA during the same period. In contrast, pupal carcasses lacking PGA tissues had static levels of DlEPV DNA and protein throughout the pupal stage. Thus, the virus population in the adult female is generated by de novo replication in her PGA, insuring that the she is equipped to successfully parasitize many hosts upon emerging. Virions in body tissues likely “seeded” the PGA early in its development.