Sequences of the reticuloendotheliosis virus (REV), an avian retrovirus, are integrated into the genome of fowl poxvirus (FPV). We developed and evaluated a quantitative multiplex real-time PCR (multiplex qPCR) assay to determine the REV-proviral load of FPV strains. Amplification efficiencies were 98.7% for the amplification of the FPV DNA and 88.7% for the amplification of REV-proviral DNA. The ratio between FPV DNA and REV-proviral DNA was calculated from the PCR efficiencies and the threshold cycle deviation of the unknown samples vs. a standard. The intraassay variation was determined by investigating triplets of different dilutions of the standard. The coefficient of variation between the threshold cycles was below 0.05 in all tested dilutions. The ratios of the triplet had a coefficient of variation of 0.201. Generally, the method overestimated the relative amount of REV-proviral DNA. Skin lesions from fowlpox outbreaks were investigated with the multiplex qPCR. The FPV:REV ratio was between 1:0.803 and 1:1.411 in samples with sufficient DNA to allow a conclusion. In addition, the investigation of cell culture material of several passages of a FPV field isolate showed a complete loss of the REV provirus after 36 passages. The loss rate of the REV provirus was approximately 50% per passage. In conclusion, we established the multiplex qPCR assay as a convenient and reliable method to determine the REV-proviral load of FPV. The first results we obtained with it show that it is of value for further investigations about the significance of the integration of the REV provirus into the genome of FPV.