Bursa samples from the United States, Mexico, and Puerto Rico were tested for the presence of infectious bursal disease virus (IBDV) using the reverse transcriptase/polymerase chain reaction-restriction endonuclease (RT/PCR-RE) assay. This assay amplifies a 394-bp fragment of the IBDV VP2 gene. A total of 151 samples were tested. Each was from a different physical location or farm. Forty-eight of the samples were determined to contain IBDV using RT/PCR. The RE profiles on 44 of these positive samples were determined using the enzymes BstNI or EcoRII, StyI, DraI, SacI, Sau3AI or MboI, and TaqI. A majority of the samples (34) had RE profiles typical of variant IBDV strains. One of six samples from Mexico was positive for IBDV. This virus had an RE profile typical of variant strains of IBDV. Three of seven samples from Puerto Rico had RE profiles characteristic of variant viruses. Two samples from the United States had RE profiles characteristic of classic vaccine IBDV strains and nine samples had new RE profiles. Five of these new profiles were BstNI- and StyI-negative, indicating that these viruses may be antigenically related to variant types. Although the new RE pattern observed in the other four samples was BstNI- and StyI-positive, it was not typical of classic vaccine IBDV strains. One flock from the United States had a mixture of two RE profiles, a typical variant type profile and an unknown variant RE profile. Two-thirds of the positive samples from flocks where the age of the birds was reported were observed between 21 and 28 days of age. The results of these studies demonstrate that the RT/PCR-RE assay can be used to diagnose IBDV in chickens and that IBDV strains exist in commercially reared chickens that have RE patterns different than known IBDV strains. The molecular differences observed using the RT/PCR-RE test were in a region of the VP2 gene, which is known to code for important neutralizing epitopes and to be highly variable among IBDV strains. Although the results demonstrate RE patterns different than those observed in known classic and variant IBDV strains, the influence of these molecular differences on biological properties of the viruses requires further investigation.