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Plantation forestry of Eucalyptus urophylla × Eucalyptus grandis supplies high-quality raw material for pulp, paper, wood, and energy and thereby reduces the pressures on native forests and their associated biodiversity. Nevertheless, owing to the heterozygosity of the E. urophylla × E. grandis genetic background, germplasm improvement by crossbreeding tends to be inefficient. As an alternative approach, genetic engineering of Eucalyptus can be used to effectively improve germplasm resources. From a strategic standpoint, increasing the plantation productivity and wood quality by transgenic technology has become increasingly important for forest industry. In this study, we established a fluorescence labelling method using CRISPR/Cas9 technology to obtain positive transformed progenies. The positive transformed progenies were easily obtained from the genetically modified population via fluorescence screening. This system can be used as a plant genome site-specific editing tool and may be useful for improving Eucalyptus genetic resources.