In the present work a real time PCR assay was developed, by using TaqMan chemistry, designing taxon-specific primers and probe able to detect Botryosphaeriaceae species, a fungal genus having its taxonomic position still under revision, that include a number of pathogens often associated to trunk canker and grapevine dieback. Study of the homology of the amplicon sequence with other fungal species was performed by the Standard nucleotide-nucleotide BLAST (blast n) of the NCBI.