A new procedure is described for the detection of Plum pox virus (PPV) in samples from large-scale field tests. The new direct real-time polymerase chain reaction (drtPCR) procedure was based on crude supernatants collected from peach (Prunus persica) leaves macerated in a buffer that was specially developed for this purpose and named “direct pathogen extract buffer.” Specific TaqMan primers and probes were designed for PPV detection: two sets specific to PPV D and M strains, respectively, and one for universal detection of PPV strains D, M, C, EA, and W. These primer and probe sets can be used singly or for duplex differentiation of D and M strains. Using Prunus spp. tissue infected with 30 known fruit tree viruses, the universal primer and probe set correctly identified PPV-infected and PPV-free samples, with no nonspecific cross-reactions. Based on endpoint analysis of the drtPCR reaction, a threshold cyle value of 36 was suggested as the maximum threshold to declare a sample positive for PPV. A comparative analysis comparing drtPCR and ELISA using 12 200 field samples revealed that drtPCR was approximately 100- to 1000-fold more sensitive than ELISA and was able to detect PPV at an earlier stage of infection than ELISA. The drtPCR is a valuable tool for PPV diagnosis and may also be applicable to other studies, including pathogen population dynamics and vector transmission efficiency.