Grapevine trunk diseases, caused by different fungal pathogens, are the most relevant group of diseases that affect vineyards, causing important economic losses. Quality control for sanitary purposes should be applied for different diseases in every stages of the plant development, from the nursery during propagation to the vineyard during grape production. Petri disease has been identified as one of the most dangerous for vine plants causing economic losses in cellars worldwide. Petri disease is caused by the infection of different fungal pathogens, mainly, Phaeomoniella chlamydospora, Phaeocremonium minimum and Cadophora luteo-olivacea. The molecular detection of plant pathogens is topical due to its facility, reproducibility and accuracy. Here we have described a PCR protocol based in a pair of new designed specific primers for the rapid detection of C. luteo-olivacea. The described procedure is intended for the direct inspection of plants suspected of being infected. Bovine serum albumin and MgCl2 were used as PCR adjuvants in order to cope with the presence of polymerase inhibitors from wood samples. Several assays have been settled out in order to assign the sensitivity and specificity of the developed technique. Under the conditions tested, the procedure has a low detection limit (10 pg) and it is specific for C. luteo-olivacea detection among other Cadophora species and fungal endophytes of Vitis vinifera.