Reticuloendotheliosis (RE) in captive greater prairie chickens (GPC, Tympanuchus cupido pinnatus) and Attwater's prairie chickens (APC, Tympanuchus cupido attwateri) was first reported in 1998. RE is caused by avian reticuloendotheliosis virus (REV), an oncogenic and immunosuppressive retrovirus infecting multiple species of wild and domestic birds. During August 2004 through May 2006 a captive population of prairie chickens was affected simultaneously with a neoplastic condition and also avian pox, the latter being detected in 7.4% (2 of 27) of all birds submitted for histopathology. A survey for REV was conducted in order to examine its possible role in mortality observed primarily in juvenile and adult specimens of prairie chickens. The investigative procedures included postmortem examinations, histopathology, molecular detection, and virus isolation. In total, 57 Attwater's prairie chickens and two greater prairie chickens were included in the study. REV infection was diagnosed using virus isolation or polymerase chain reaction (PCR) or both in 59.5% (28 of 47) of blood samples and/or tumors from suspect birds. Lymphosarcomas were detected in the tissues of 37% (10 of 27) of the birds submitted for histopathology. Such lymphosarcomas suggestive of RE represented the most frequent morphologic diagnosis on histopathology among 27 separate submissions of naturally dead prairie chickens. Overall, REV was detected or RE diagnosed in 34 of 59 prairie chickens (57.62%). The average death age of all birds diagnosed with lymphosarcomas on histopathology was 2.2 yr, ranging from <1 to 4 yr. Although deaths associated with neoplasia occurred in males and females in equal proportions based on submissions, overall more males were diagnosed as REV infected or RE affected (16 males vs. 7 females, and 11 birds of undetermined gender). Reticuloendotheliosis virus was confirmed as a significant cause of mortality in captive prairie chickens.