Woody canker diseases caused by fungi of theBotryosphaeriaceaefamily are producing increasing losses in many economically important woody crops, including almond. To develop a molecular tool for the detection and quantification of the most aggressive and threatening species is of main importance. This will help to prevent the introduction of these pathogens in new orchards and to conveniently apply the appropriate control measures. Three reliable, sensitive and specific duplex qPCR assays using TaqMan probes have been designed for the detection and quantification of (a)Neofusicoccum parvumand theNeofusicoccumgenus, (b)N. parvumand theBotryosphaeriaceaefamily and (c)Botryosphaeria dothideaand theBotryosphaeriaceaefamily. The multiplex qPCR protocols have been validated on artificially and naturally infected plants. Direct systems to process plant materials, without DNA purification, allowed high-throughput detection ofBotryosphaeriaceaetargets even in asymptomatic tissues. These results validate the qPCR using the direct sample preparation method as a valuable tool for Botryosphaeria dieback diagnosis allowing a large-scale analysis and the preventive detection of latent infection.