Populus × euramericana (Dode) Guinier clone (cl.) “I-214” is a fast-growing interspecific hybrid between Eastern cottonwood (P. deltoides Bartr. ex Marsh) and European black poplar (Populus nigra L.). Populus × euramericana was introduced into Serbia in the 1950s and has become one of the most widely grown poplar species. In September 2019, cankers were observed on stems and branches of P. × euramericana cl. “I-214” trees in a two-year-old poplar plantation in the province of Vojvodina, Serbia. The canker tissue was soft and watery, and a colorless fluid that smelled rotten flowed from the cracks in the bark, suggesting possible bacterial disease. After two weeks, diseased trees experienced crown die-back and oozing of foamy, odorous exudates and this study aimed to identify the causal agent of the disease. Canker margins and exudates were collected from 20 symptomatic trees. The associated bacterium was isolated and identified using biochemical characteristics, phylogenetic analyses based on 16S rRNA gene sequences, and multilocus sequence analyses (MLSA) based on partial sequencing of three housekeeping genes (gyrB, infB, and atpD). The pathogen was identified as Lonsdalea populi. Pathogenicity tests were conducted on rooted cuttings of P. × euramericana cl. “I-214” in an environmental test chamber and demonstrated that the isolated bacterial strain was able to reproduce symptoms of softened, water-soaked cankers and exudation. To the best of our knowledge, this is the first report of L. populi causing bacterial canker disease on P. × euramericana cl. “I-214” in Serbia and in southeastern Europe (SEE). It is also the first report of a bacterial disease on hybrid poplars, including P. × euramericana in this country and in SEE. If the disease spreads into new areas, selection for L. populi resistance may need to be integrated into future poplar breeding programs.