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The aim of this study was to investigate the molecular identification and assess the genetic relationship of baculovirus isolated from Southern Vietnam. The diseased insect samples were collected from the different fields. The partial sequence of 450 base pairs of lef-8 gene was amplified and sequenced to assess the genetic variations of baculovirus isolates specific for Spodoptera litura, Helicoverpa zea, and Helicoverpa armigera. The sequences alignment demonstrated that Helicoverpa zea specific isolates exhibited six single nucleotide polymorphic sites. Whereas, twenty five single polymorphic sites were found in Spodoptera litura specific isolates. Thus, Spodoptera litura specific isolates were higher polymorphic than Helicoverpa zea specific isolates. The genetic distance analyses showed that the distance between Vietnamese baculovirus isolates and Group II Alphabaculovirus isolates was lower than other Baculovirus groups. The phylogeny of Vietnamese isolates in relation to other baculovirus isolates was also determined using partial sequences of lef-8 gene. The phylogenetic tree placed all Vietnamese isolates in Group II Alphabaculovirus, where seven Vietnamese Helicoverpa zea specific isolates were most closely related to Helicoverpa zea SNPV, fourteen Vietnamese Spodoptera litura specific isolates were located with Spodoptera litura NPV-G2 in one clade and a Vietnamese Helicoverpa armigera isolate was appeared to be closely related to Helicoverpa armigera SNPV-NNg1, Helicoverpa armigera NPV-C1, Helicoverpa armigera NPV-G4.