A total of 189 nucleopolyhedrovirus (NPV) clones were obtained from the NPV-infected larvae of the common cutworm, Spodoptera litura, collected in Japan. Restriction endonuclease (REN) analysis demonstrated that these 189 S. litura NPV (SpltNPV) clones exhibited 33 distinct REN patterns that represented three distinct NPV types designated as type A, type B, and type C SpltNPVs based on the similarity of overall REN patterns. Comparative examination of REN patterns from these clones in this study with those from NPVs published previously indicated that type A SpltNPV corresponded to Spodoptera littoralis NPV (SlNPV)-D or SlNPV-B, whereas type B SpltNPV corresponded to the NPVs widely identified in S. litura larvae in Japan, China, and the Philippines. The overall REN pattern of type C SpltNPV did not resemble REN patterns from any of those NPVs previously identified in S. litura and S. littoralis and was distinct from the REN patterns of NPVs from Autographa californica, Bombyx mori, Hyphantria cunea, Spodoptera exigua, Lymantria dispar, and Orgyia pseudotsugata, suggesting that type C SpltNPV was a novel NPV pathogenic for S. litura. Comparative characterization with S. litura cultured cells and larvae showed that the biological properties with respect to productivity and virulence differed significantly not only among the types of SpltNPV but also among the variants within a SpltNPV type, demonstrating that SpltNPV-C3, a clone from type C SpltNPV, caused rapid mortality of S. litura larvae compared with those clones of type A or type B SpltNPV. These results indicate that the natural heterogeneity of baculoviruses can be exploited to develop NPV strains suitable for the control of S. litura and suggest that the novel SpltNPV-C3 isolated in this study has high potential to be developed as an effective pest control agent for S. litura.