Nine reticuloendotheliosis virus (REV) isolates obtained from broiler breeders, turkeys, and prairie chickens located in three different geographical regions in the USA, and three isolates obtained from known contaminated live-virus vaccines were characterized using polymerase chain reaction (PCR) and indirect immunofluorescence (IFA) assays. All isolates were propagated in chicken embryo fibroblasts obtained from a specific pathogen free breeder flock. PCR analysis of all 12 isolates resulted in the amplification of the 291-bp REV long-terminal repeat region (LTR); none of the isolates exhibited a different pattern or shift from the expected PCR product of REV LTR. The subtype of the REV isolates was determined by IFA using REV-specific monoclonal antibodies, 11B118.22, 11C237.8, and 11D182. Results from sub-typing indicated that all nine isolates from broiler breeders, turkeys, and prairie chickens belonged to subtype 3, and are antigenically related to the chick syncytial virus (CSV) strain of REV, the prototype of subtype 3 REV. In contrast, the three isolates from contaminated vaccines were classified as subtype 2, and were antigenically related to spleen necrosis virus (SNV) strain of REV, the prototype of subtype 2 REV. Three isolates representing REV isolated from broiler breeders, turkeys, and prairie chickens were cloned and further evaluated by DNA sequence analysis of the envelope gene. Results from DNA sequence analysis confirmed those from sub-typing and indicated that the three REV isolates representing those from broiler breeders, turkeys, and prairie chickens are closely related to CSV of REV, with an amino acid homology of 98% or greater as compared with SNV with an amino acid homology of 95% or less. Data from this study clearly indicate that subtype 3 is the most common subtype of REV circulating in three different avian species, namely broiler breeders, turkeys and prairie chickens, located in three different geographical regions in the United States.