The Grapevine Trunk Diseases (GTDs) are the most common diseases of grapevine wood inducing a slow decay leading to plant death. Due to the environmental impact, chemical treatments are no longer authorized, and prevention or trunk removal are the last available control methods. Fighting against these slow evolving diseases requires a better knowledge of fungal and bacterial communities associated with GTDs. Our approach is based on fungal species identification using ITS (Internal Transcribed Spacer) sequences obtained by pyrosequencing (Roche 454) of grapevine wood samples. DNAs were extracted from different parts of grapevine trunks and amplified using fungal specific ITS primers. A workflow was set up to analyze pyrosequencing data, allowing taxonomic assignment with a database extracted from Genbank and curated with the FungalITSextractor (Nilsson H et al. 2010). The pipeline links tools, including cleaning and extracting ITS sequences to limit the impacts of sequencing errors on clustering and assignation steps. Then, Operational Taxonomic Units (OTUs) detection and taxonomic assignments were performed with the QIIME package (Caporaso JG et al. 2010). Samples from different vineyards (infected or not), with several dates of sampling, were analyzed. Different ITS PCR primers and technical replicates were tested using controls corresponding to the mixture of fungal DNAs from diverse known species. These controls highlighted interests and limits of PCR amplicons pyrosequencing and the relevance of the bioinformatics methods to extract accurate data to fit to the context of taxonomy.