Seven Gram-negative bacterial strains were isolated from oozing bark canker of poplar (Populus × euramericana) trees in Hungary. They showed high (>98.3%) 16S rRNA gene sequence similarity to Lonsdalea quercina; however, they differed from this species in several phenotypic characteristics. Multilocus sequence analysis based on three housekeeping genes (gyrB, atpD and infB) revealed, and DNA-DNA hybridization analysis confirmed, that this group of bacterial strains forms a distinct lineage within the species Lonsdalea quercina. A detailed study of phenotypic and physiological characteristics confirmed the separation of isolates from poplars from other subspecies of L. quercina; therefore, a novel subspecies, Lonsdalea quercina subsp. populi, type strain NY060(T) (=DSM 25466(T)=NCAIM B 02483(T)), is proposed.