Two genetically distinct Spodoptera litura nucleopolyhedrovirus (SpltNPVs) isolates from Pakistan were investigated by deep sequencing of their full genomes. Previously we reported that isolate SpltNPV-Pak-TAX1 kills S. litura (leafworm) larvae significantly faster than SpltNPV-Pak-BNG. Here we find that the consensus sequences of these two virus isolates shared 99% identity, suggesting they are closely related. The major difference between these two isolates is the absence of hr17 (putative enhancer of transcription and origin of replication) in SpltNPV-Pak-TAX1 and the absence of ORF125 with unknown function in SpltNPV-Pak-BNG. Analysis of the rates of nonsynonymous and synonymous single-nucleotide substitutions showed that strong purifying selection predominates, although for a small number of genes there was neutral or positive selection. The most striking case is ORF122, which encodes a putative viral fibroblast growth factor (FGF), known to be involved in the passage of virus from the midgut to the interior of the larva and linked to virus virulence in other baculoviruses. We found very little polymorphism within both virus isolates, a result at odds with observations for other baculoviruses, possibly suggesting recent dispersal of the virus in Pakistan. We have therefore identified two loci possibly linked to the enhanced virulence of the SpltNPV-Pak-TAX1. This information could help to understand the enhanced activity of SpltNPV-Pak-TAX1 and to select better SpltNPV isolates for the control of S. litura in Pakistan and elsewhere.