Abstract BACKGROUND: To assess the persistence of genetically modified and naturally occurring baculoviruses in an aquatic environment, replicate (three) outdoor, aquatic microcosms were spiked with spruce budworm viruses [Ireland strain of Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) and the recombinant CfMNPVegt − /lacZ + ] at a rate of 1.86 × 10 10 occlusion bodies (OBs) m −2 of surface area. The presence of virus in water samples collected at various times after inoculation was determined by PCR amplification of baculoviral DNA extracted from OBs. RESULTS: Although UV radiation rapidly degrades baculoviruses under natural conditions, both viruses persisted above the level of detection (>100 OBs 450 µL −1 of natural pond water) for at least 1 year post‐inoculation, with little difference between the viruses in their patterns of persistence. CONCLUSION: The present microcosm study suggests that occlusion bodies of baculoviruses can persist in the flocculent layer of natural ponds. On disturbance, OBs could re‐enter the main water column and thus be available for transport to new locations. Implications for environmental risk assessment are discussed. © 2008 Her Majesty the Queen in Right of Canada. Canadian Forestry Service. Published by John Wiley & Ltd.