A sensitive diagnostic assay for the detection of infections with the bovine lentivirus Jembrana disease virus (JDV) is required in Indonesia to control the spread of Jembrana disease. Immunoassays are used routinely but are compromised by cross-reactive epitopes in the capsid (CA) protein of JDV and the genetically related bovine immunodeficiency virus (BIV). JDV gag-specific primers were tested in a real-time PCR assay to detect proviral DNA in peripheral blood mononuclear cells from 165 cattle from the Tabanan district of Bali. JDV-specific amplicons were detected in 9% of the cattle and only 33% of the real-time PCR positive cattle were seropositive. The delayed seroconversion that occurs after infection with JDV could explain the low concordance between these assays but other factors may be responsible. BIV proviral DNA was not detected in any of the PBMC DNA samples. A high concordance value of 98.6% was found between the JDV plasma-derived antigen Western blot and the JDV p26-his recombinant protein ELISA. Only 21% of the seropositive cattle had detectable levels of proviral DNA suggesting that the proviral load in recovered cattle is low. A combination of real-time PCR and JDV p26-his ELISA is recommended for the detection of infection with JDV in Indonesia.