Eutypa lata is the causal fungal agent of Eutypa dieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detect E. lata in grapevine wood samples. We designed six pairs of PCR primers for diagnosis of E. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of the E. lata isolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identify E. lata from pure or mixed cultures as well as from grapevine wood samples. Identification of E. lata in wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases.