Accurate differentiation of the pathogenic phenotypes of infectious bursal disease viruses (IBDVs) will instruct effective vaccination programs and improve the study of the molecular epidemiology of IBDVs. In this study, an 833 bp hypervariable nucleotide region was identified in VP2 genes of known IBDVs with different virulences through multiple sequence alignment. Moreover, using NEBcutter software analysis, two restriction enzyme sites, SpeⅠ(generating 531 and 302 bp fragments) and StuⅠ(generating 242 and 591 bp fragments) were found presented in very virulent but not attenuated IBDVs. Moreover, the restriction enzyme site SacⅠ(generating 218 and 615 bp fragments) presented in attenuated IBDVs but not very virulent IBDVs. Therefore, a reverse-transcription (RT)-PCR combined with a restriction fragment length polymorphism (RFLP) assay was developed to differentiate attenuated and very virulent IBDVs. The RT- PCR assay was used to confirm 282 IBDV positive samples from 310 suspicious dead chicken samples. The 60 IBDV positive samples were used to evaluate the assay, followed by confirmation via gene sequencing and histopathological examinations of the bursas of Fabricius from chickens infected by these IBDVs. The results showed that 24 viral strains with SpeⅠand StuⅠsites were very virulent, causing severe pathological damage in the bursas of Fabricius, while 36 viral strains with the SacⅠsite were attenuated IBDVs, exhibiting only slight pathological damage. The combined RT-PCR and RFLP assay provided a useful approach for differentiating the pathogenic phenotypes of IBDVs.