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<i>Marssonina brunnea</i> is the main pathogen that causes poplar black spot disease, which leads to the decrease of the photosynthetic efficiency and significantly affects the production and quality of timber. Currently, no in-field diagnostic exists for <i>M. brunnea</i>. Here, we described a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of <i>M. brunnea</i>. A set of six oligonucleotide primers was designed to recognize eight distinct sequences of the internal transcribed spacer (<i>ITS</i>) region of <i>M. brunnea</i>. The LAMP assay was optimized by the combination of high specificity, sensitivity, and rapidity for the detection of less than 10 pg/μL of target genomic DNA in 60 min per reaction at 65 °C, whereas with PCR, there was no amplification of DNA with concentration less than 1 ng/μL. Among the genomic DNA of 20 fungalisolates, only the samples containing the genomic DNA of <i>M. brunnea</i> changed from violet to sky blue (visible to the naked eye) by using hydroxynaphthol blue (HNB) dye. No DNA was amplified from the eight other fungus species, including two other <i>Marssonina</i> pathogens, three other foliar fungi pathogens of poplar, and three common foliar fungal endophytes of poplar. Moreover, the detection rates of <i>M. brunnea</i> from artificially and naturally infected poplar leaves were 10/16 (62.5%) and 6/16 (37.5%) using PCR, respectively, while the positive-sample ratios were both 16/16 (100%) using the LAMP assay. Overall, the <i>ITS</i> LAMP assay established here can be a better alternative to PCR-based techniques for the specific and sensitive detection of <i>M. brunnea</i> in poplar endemic areas with resource-limited settings.