Ovine pulmonary adenocarcinoma (OPA) is a chronic respiratory disease with long incubation period, caused by Jaagsiekte sheep retrovirus (JSRV). The study was performed to evaluation TaqMan real-time PCR technique, as a highly sensitive and specific method, to investigate JSRV proviral DNA in whole blood samples from sheep with chronic pneumonia and apparently healthy cases in a slaughterhouse survey. Blood samples were taken via jugular venipuncture of 80 sheep with chronic pneumonia and 20 apparently healthy sheep. After slaughtering, lungs were carefully examined then tissue samples from ten fix locations of each lung and caudal mediastinal lymph node were collected for comparison real-time PCR assay results’ to those of histopathological examinations. The blood samples were stored at -80 °C until DNA extraction. DNA samples were subjected to real-time PCR to investigate JSRV proviral DNA. Fifteen out of eighty blood samples (18.75%) from sheep affected by chronic pneumonia and three blood samples out of twenty apparently healthy sheep (15%) were found to be positive for JSRV proviral DNA by TaqMan real-time PCR. Moreover, 11 out of 80 (13.75%) corresponding lung samples from sheep affected by chronic pneumonia and 2 out of 20 (10%) corresponding lung samples from apparently healthy sheep showed microscopic changes consistent with OPA. Chi square test didn’t show significant difference in the health status and real-time PCR (p=0.69) or histopathological findings (p=0.66). However, it showed that there was high relationship between the results of real-time PCR and histopathological assays (p≤ 0.01). Therefore, the present study confirmed that real-time PCR is a sensitive and specific test for accurate detection of JSRV infection in both pneumonic and apparently healthy sheep.