Abstract:[Objective] To study the epidemiological status of reticuloendotheliosis virus (REV) infection in ducks. [Methods] Two hundred and twenty tissue samples of the Bursa, spleens and livers were randomly collected from 97 sick or dead ducks from 3 areas in Shandong Province, China. REV infections were tested by virus isolation in cell cultures, direct dot blot hybridization, regular PCR and nest-PCR with tissue-extracted DNA as the templates. PCR products of env gene fragments were cloned and sequenced, sequences of env gene fragments were compared and analyzed. [Results] REV infections were detected in 35/39 (90%) bursal samples, 54/84 (64%) spleens and 32/97 (33%) livers by Nest-PCR. The positive rate of bursal samples was significantly higher than spleen and liver samples (P<0.01). No positive sample was detected by virus isolation, direct dot blot hybridization or regular PCR from the same Bursa, spleen and liver samples. In sequence comparisons of amplified env gene fragments, Yinan-1 and Binzhou-1 had 99.8% of identity with reference SNV strain isolated from ducks in USA, and Linqu-1 had 100% identity with two other reference strains isolated from chickens in USA. However, they had lower homology with strains isolated from chickens in China. [Conclusion] More attention should be given to bursal samples in detection of REV. Since REV was existing only at a very low level in infected sam-ples and difficult to be detected, nest-PCR should be conducted as the more sensitive assay. Sequence homology com-parisons and phylogenetic tree analysis suggested that REV infections in these ducks may originally come from imported breeder ducklings without inspection for REV.