A technique using the polymerase chain reaction (PCR) was developed for detection of the nucleopolyhedrovirus (NPV) polyhedrin gene. 152 nucleotide sequences of polyhedrin gene were compared in pairwise and multiple alignment sequences. Eleven highly conserved DNA sequences within the coding region of the polyhedrin gene were identified. Two candidate regions were targeted for amplification and consequently one pair of degenerate PCR primers was designed to produce fragments of about 355 bp. The NPVs tested by this technique were Autographa californica (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea NPV (HcNPV), Lymantria dispar NPV (LdNPV), Spodoptera exigua NPV (SeNPV), S. litura NPV (SlNPV), Spodoptera littoralis NPV (SpliNPV) and nine local NPV isolates. Furthermore, three randomly chosen PCR products were cloned and sequenced. The sequencing data showed that the three PCR products were fragments of polyhedrin gene. Conclusively, this technique would be useful in monitoring the environmental fate, distribution of NPVs, release of the wild type and recombinant NPVs and quality control studies of baculoviral insecticides as well.