BACKGROUND: Tubulin proteins, the main components of microtubules in all eukaryotes, are involved in numerous aspects of plant morphogenesis and adaptation to the environment. In woody plants, microtubules are closely associated with the orientation of cellulose microfibril deposition in the secondary xylem cells and thereby exert an influence on the strength and flexibility of wood. Three major types of tubulin proteins-α-, β- and γ-tubulin-are ubiquitously present in all flowering plants, with α- and β- tubulin serving as basic subunits of microtubules and γ-tubulin directing microtubule nucleation. Compared with herbaceous plants, information on tubulin gene family has been limited in forest trees. This study aimed to characterize the tubulin gene family in the model forest tree Populus deltoides. RESULT: Based on the whole genome sequence of P. deltoides, 25 PdTubulin genes were identified, including 6 PdTUAs, 17 PdTUBs, and 2 PdTUBGs were identified, with an uneven distribution across 14 chromosomes. Unlike Arabidopsis, which has only three pairs of tubulin paralogs, nearly all PdTubulin were paralogous duplicates, primarily generated by p-whole genome duplication (WGD), γ-WGD, or segmental duplication, indicating multiple rounds of gene family expansion. After the duplication events, the number of TUA genes in Populus was more strictly constrained compared to TUB genes. All paralogous and orthologous tubulin pairs have been under strong purifying selection. Expression analysis revealed that each PdTubulin gene was preferentially expressed in one of three organs: root, leaf, or stem. 5 PdTUB paralogs exhibited similar expression patterns, suggesting potential redundancy. Additionally, expression analysis in male and female floral buds across developmental stages indicated that different members might be involved in sex-specific processes.