A real-time loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Botrytis cinerea in grapevine bunch trash, immature berries, and ripening berries. A simple method for the preparation of crude extracts of grape tissue was also developed for on-site LAMP analysis. When tested with 14 other fungal species frequently found in grapevines, the LAMP assay was specific and sensitive to a B. cinerea DNA quantity of 0.1 ng/µL. The sensitivity was further tested using bunch trash samples with B. cinerea colonization levels between 6 and 100% and with bulk-berry samples composed of 4 pathogen-free berries or 4 berries among which 25 to 100% had been inoculated with B. cinerea. The LAMP assay detected the lowest B. cinerea colonization level tested in bunch trash and in immature and mature berries in less than 20 min. In single-berry experiments, LAMP amplified B. cinerea DNA from all artificially inoculated individual immature and mature berries. No amplification occurred in B. cinerea-free material. The real-time LAMP assay has the potential to be used as a rapid on-site diagnostic tool for assessing B. cinerea colonization in bunch trash and B. cinerea latent infections in berries, which represent critical stages for decision-making about disease management.