Aims: This paper describes a quick, reproducible, sensitive method for baculoviral DNA extraction, purification and detection from freshwater and forest litter environments. Methods and Results: The extraction protocol utilizes enzymatic and chemical lysis and physical disruption. To assess the efficiency of the extraction and purification protocol, PCR was used to detect a 530 bp DNA fragment from the genome of a genetically-modified baculovirus, Choristoneura fumiferana NPVegt(-)/lacZ(+). The detection limit of PCR amplification nias routinely about 4.1 x 10(2) occlusion bodies (OBs) 450 mul(-1) lake water. Template DNA from the detritus and forest litter samples required 100-fold dilutions before use in PCR reactions. The detection limits for detritus and forest litter samples were routinely about 7.41 x 10(3) and 2.08 x 10(4) OBs 0.5 g(-1) dry weight, respectively. Conclusions: The DNA extraction and purification methodology is reproducible, sensitive and can be used in lieu of, or in conjunction with, insect bioassays. Significance and Impact of the Study: The DNA extraction and purification protocol described in this paper will facilitate risk assessment and ecological studies of both wild-type and genetically-modified baculoviruses.