β-d-xylosidase (BXL) hydrolyzes xylobiose and xylo-oligosaccharides into xylose monomers, and is a rate-limiting enzyme in the degradation of hemicellulose in the cell wall. In this study, ten genes encoding putative BXL proteins were identified in the Populus trichocarpa genome by bioinformatics methods. In the phylogenetic analysis, the PtBXLs formed two subfamilies. PtBXL8 and PtBXL9 were closely related to AtBXL1, an important enzyme in the normal development of the Arabidopsis cell wall structure. Chromosomal distribution and genome synteny analyses revealed two tandem-duplicated gene pairs PtBXL3/4 and PtBXL6/7 on chromosomes II and V, respectively, and six segmental-duplicated gene pairs on chromosomes II, V, VIII, X, and XIV among the PtBXL gene family. Tissue-specific expression data from PlantGenIE indicated that PtBXL2, 4, 5, and 10 were highly expressed in stems. Quantitative real-time RT-PCR analyses revealed that PtBXL4, 5, and 9 were up-regulated in the upper stem in response to the low and high ammonium and nitrate treatments. The influence of nitrogen on the expression of PtBXL4, 5, and 9 genes may affect the formation of the plant secondary cell wall. This comprehensive analysis of the BXL family in poplar provides new insights into their regulation by nitrogen and increases our understanding of the roles of BXLs in hemicellulose metabolism in the secondary cell wall and during plant development.,© King Abdulaziz City for Science and Technology 2020.