The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.