The genome of Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) was completely sequenced and compared with whole genome sequences of the Agrotis segetum nucleopolyhedrovirus A (AgseNPV-A) and Agrotis ipsilon nucleopolyhedrovirus (AgipNPV). The AgseNPV-B genome is 148,981 bp in length and encodes 150 putative open reading frames. AgseNPV-B contains two copies of the gene viral enhancing factor ( vef ), making the Agrotis nucleopolyhedroviruses and A. segetum granulovirus (AgseGV) very rich in vef in comparison to other baculoviruses. Genome alignments of AgseNPV-B, AgseNPV-A and AgipNPV showed a very high genome co-linearity interspersed with variable regions, which are considered as putative sites of genomic recombination. Phylogenetic analyses revealed that all three viruses are distinct. However, AgseNPV-B is more closely related to AgipNPV suggesting that both viruses are at an early stage of phylogenetic divergence. It is proposed that AgseNPV-B belongs to a third Alphabaculovirus species of the Agrotis baculovirus complex. The Agrotis exclamationis nucleopolyhedrovirus (AgexNPV) shared high nucleotide sequence identities with AgseNPV-B, suggesting it is actually an AgseNPV-B isolate.