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The need to increase food production together with the demand to reduce the application of synthetic chemicals that have consequences on increasing the carbon footprint and negative impacts on the environment and human health, led to the search for alternative methods to protect plants against pathogens. The use of the highly sensitive real-time quantitative PCR (qPCR) arises as an extremely useful tool for studying various agents of infection in plants, such as fungi, viruses, or bacteria leading to a better control of diseases and limiting the use of chemical defence strategies. Given the high incidence of diseases in tomato plants caused by Fusarium spp., their consequent negative economic impacts, and the fact that most phytosanitary treatments are based on the application of synthetic fungicides, the establishment of a molecular-based tool that enables their early and accurate detection is of great interest. Furthermore, it will provide an additional tool for the screening of resistant plants. In the presented study, a TaqMan®-based qPCR method targeting the Fusarium spp.- specific internal transcribed spacer (ITS) region was developed for the simultaneous detection and quantification of a panoply of Fusarium species that affect tomato. As a proof of principle, the new qPCR assay was used to assess Fusarium spp. contamination of tomato field plants and of plants grown under controlled conditions. qPCR combined with the chemistry of TaqMan® MGB probes represents a highly specific and sensitive detection system, even when low amounts of target DNA are present, as in the case of early plant–fungi interactions.