The virulence determination of four Euproctis pseudoconspersa nuclear polyhedrosis virus (EpNPV) isolates showed that Zhejiang isolate is the most toxic one (LC₅₀=2.50×10⁶PIB/mL), and Hunan isolate is the weakest isolate (LC₅₀=13.36×10⁶PIB/mL). The virulence of different isolates in order is Zhejiang (ZJ), Guizhou (GZ), Hubei (HB) and Hunan (HN), besides GZ and HB isolates have similar virulence, which LC₅₀ were 9.50×10⁶PIB/mL and 7.31×10⁶PIB/mL respectively. Five genes of the four isolates about virus reproduction, interaction between virus and host and virulence were determined and sequenced, genetic structure based on these genes among isolates was analysed, the result indicated the similarity of HB and GZ, HN are highest (99.7%), and that of ZJ between HN isolate are lowest (99.4%). The NJ and UPGMA phylogenetic trees based on five genes also showed that HB and HN isolates first clustered into one group, then clustered with GZ, finally clustered with ZJ isolate, which indicated that the genetic distance among ZJ and other three isolates are relatively far. Both virulence determination and genetic structure analysis suggested virulence and heredity among four virus isolates are closely related.