Abstract: In order to separate and purify the main pathogen bacteria to wet-heartwood disease in Populus and the correspondent antagonist bacteria, dominant strains were separated and purified from pathogenic tissues of different Populus varieties, then tested the pathogenicities by inoculation to sterile Populus seedlings. The pathogen and correspondent antagonist bacteria were classified and identified by morphology, physiological-biochemical characteristics, fatty acid composition analysis, 16SrRNA sequence and MLSA. The results show that 10 dominant strains are separated and purified, and pathogen 12-1 caused evident symptom after inoculation to sterile poplar seedlings, and it is classified and identified as Brenneria salicis; Eighteen endogenous strains are isolated from the stem of Populus trees, of which J-4 isolate is selected and verified as antagonist bacteria of the pathogen bacteria strain 12-1 by the confrontation culture and the inoculation in Populus sterile seedlings, and it is preliminary determined to be Paenibacillus taiwanensis. In conclusion, Brenneria salicis is the main pathogen to wet-heartwood disease in Populus, and Paenibacillus taiwanensis ias the correspondent antagonist bacteria.