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Abstract Background Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) is considered a promising biocontrol agent that can be used for the effective control of the cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), which is an economic pest on many cultivated plants and crops in tropical and sub-tropical regions. The genome of the SpliNPV-AN1956 isolate has been fully sequenced, providing a reference strain for comparison of new isolates. In this study, identification, biological activity, and genetic characterization of a field collected SpliNPV isolate were carried out. Results The examination of viral occlusion bodies (OBs) by TEM showed a typical baculovirus OBs of type nucleopolyhedrovirus (NPV) with polyhedral structures. The phylogenetic analysis of the late expression factor- 5 (lef-5) gene as well as the restriction profile analysis confirmed the identity of SpliNPV as a variant isolate. Digestion with ScaI endonuclease showed that 4 fragments of 50, 35, 27, and 13 kb were detected but 3.2 kb was absent in SpliNPV-Cab3 pattern compared to the reference strain. Meanwhile, upon digestion with PstI and HindIII endonucleases, no differences were observed in both isolates’ pattern. Moreover, the virulence evaluation against S. littoralis 1st instar larvae indicated that LC50 value of SpliMNPV-Cab3 was higher than that estimated for the reference strain. Also, ST50 of SpliNPV-Cab3 (120 h) was significantly different with that of the reference strain (132 h). Conclusion The host specificity based on virulence parameters make SpliNPV-Cab3 isolate a potential candidate to be involved in the integrated pest management strategies for the control of S. littoralis population with a prospect to biopesticide development.