A baculovirus has been isolated from infected larvae of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae) in Taiwan. Ultrastructural observation revealed that this virus is a multiple nucleopolyhedrovirus (MNPV), and the name L. xylina MNPV (LyxyMNPV) was proposed. Restriction endonuclease (BamHI, EcoRI, and EcoRV) profiles of LyxyMNPV genome differed from those of other known NPVs. The size of the LyxyMNPV genome was estimated to be 154+/-1.26 kbp (mean+/-SE). The polyhedrin gene is located in the BamHI-D, EcoRI-C, and EcoRV-K fragments of LyxyMNPV genome. The gene organization of the LyxyMNPV EcoRV-K fragment and the phylogenetic analysis based on the polyhedrin gene sequences showed that LyxyMNPV is closely related to the L. dispar MNPV (LdMNPV). Furthermore, a rapid assay method was developed to distinguish LyxyMNPV from LdMNPV based on PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis of polyhedrin genes.