Five isolates from The United States (SC1 isolated from H. virescens, SC2 from H. zea, Sf82-126 from S. frugiperda, SC3 from H. virescens and SC4 from S. frugiperda) and two isolates from Indonesia (INDO1 and INDO2 from S. exigua) were compared with respect to the general relatedness of their DNA sequence. Restriction enzyme analysis revealed that INDO1 and INDO2 had similar REN profiles which differed significantly from all other isolates. However, there were noticeable differences between the two isolates. INDO1 and INDO2 are also closely related to SeAV-5a. Isolate SC1 is similar to HvAV-3a. Comparison with isolates from T. ni shows that SC2, SC3 and Sf82-126 are very similar to TnAV-2a. Isolate SC4 is identical to SfAV-1a. Southern blot analysis showed that INDO2 is identical with SeAV-5a and closely related to INDO1; SC4 is identical to SfAV-1a. Western blot analysis of ascovirus protein using SeAV-5a antibody confirmed that INDO1, INDO2 and SC1 are related to SeAV-5a. SeAV-5a p50 antibody also hybridized to SC4. Phylogenetic trees generated from DNA polymerase and P50 gene sequences showed that ascovirus isolates used in this study are grouped into four known and described ascovirus species, SfAV-1a, TnAV-2a, HvAV-3a and SeAV-5a.