An emerging poplar canker caused by the gram-negative bacterium, Lonsdalea populi, has led to high mortality of hybrid poplars Populus × euramericana in China and Europe. The molecular bases of pathogenicity and bark adaptation of L. populi have become a focus of recent research. This study revealed the whole genome sequence and identified putative virulence factors of L. populi. A high-quality L. populi genome sequence was assembled de novo, with a genome size of 3,859,707 bp, containing approximately 3434 genes and 107 RNAs (75 tRNA, 22 rRNA, and 10 ncRNA). The L. populi genome contained 380 virulence-associated genes, mainly encoding for adhesion, extracellular enzymes, secretory systems, and two-component transduction systems. The genome had 110 carbohydrate-active enzyme (CAZy)-coding genes and putative secreted proteins. The antibiotic-resistance database annotation listed that L. populi was resistant to penicillin, fluoroquinolone, and kasugamycin. Analysis of comparative genomics found that L. populi exhibited the highest homology with the L. britannica genome and L. populi encompassed 1905 specific genes, 1769 dispensable genes, and 1381 conserved genes, suggesting high evolutionary diversity and genomic plasticity. Moreover, the pan genome analysis revealed that the N-5-1 genome is an open genome. These findings provide important resources for understanding the molecular basis of the pathogenicity and biology of L. populi and the poplar-bacterium interaction.