Several extraction and purification techniques were evaluated on recovery of free baculoviral DNA from pond water and sediment samples. The best methodology was selected based detection sensitivity and reproducibility. A preliminary laboratory experiment looking at the persistence of baculoviral DNA in aquatic microcosms was performed. Results showed that baculoviral DNA persisted in non-autoclaved pond water and sediment for at least 7 days and 1 day, respectively. A field aquatic microcosm experiment was then conducted to investigate baculoviral DNA persistence in pond water and sediment in a northern temperate forest environment. Target DNA was detected for a period of up to 24 hrs in pond water. Persistence in sediment was not observed as all samples were negative for detection of the target fragment. It is likely that nuclease degradation and binding to sediment particles resulted in detection difficulty in sediment samples. Quantification of dsDNA in pond water and sediment sample extracts using PicoGreen® indicated a correlation between total dsDNA concentrations recovered from field samples and successful PCR detection of a target 530bp baculoviral DNA fragment during the observed period of persistence.