Infectious bursal disease viruses (IBDVs) in 26 IBDV-positive bursa samples collected in Croatia during the period 1996–2000 and in two commercially available vaccines were differentiated by the presence or absence of the CfoI, SacI, SspI, StuI, and TaqI restriction sites in the 422-bp fragment of segment A of the VP2 gene (nt 732–1153). The fragments from 14 (54%) field isolates were TaqI+ StuI+ SspI+ and SacI− CfoI−, indicating their very virulent (vv) character. The presence of CfoI restriction site in 10 (38%) field isolates is uncommon for vvIBDV strains. It was detected in only the 88180 vvIBDV strain. Nevertheless, these isolates can be classified as vv strains according to TaqI+ StuI+ SspI+ SacI− restrictions. Two SacI+ StuI+ CfoI+ TaqI− SspI− field isolates (8%) could be classified as non-vvIBDVs. The StuI+ restriction is common to vvIBDV strains. However, the StuI recognition sequence is present in the F52/70 classic European and 002-73 attenuated strains as well. The SacI+ CfoI+ StuI− SspI− restrictions and the lack of the TaqI restriction at nt position 832 show that the IBDV in GUMBOKAL® IM-SPF vaccine corresponds to the attenuated and/or vaccine strains. The TaqI restriction at nt position 875 suggests that the IBDV in GUMBOKAL® SPF vaccine could belong to the mild strains.