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The <i>BTB</i> (Broad-complex, tramtrack, and bric-a-brac) gene family, characterized by a highly conserved BTB domain, is implicated in a spectrum of biological processes, encompassing growth and development, as well as stress responses. Characterization and functional studies of <i>BTB</i> genes in poplar are still limited, especially regarding their response to hormones and biotic/abiotic stresses. In this study, we conducted an HMMER search in conjunction with BLASTp and identified 95 <i>BTB</i> gene models in <i>Populus trichocarpa</i>. Through domain motif and phylogenetic relationship analyses, these proteins were classified into eight families, NPH3, TAZ, Ankyrin, only BTB, BACK, Armadillo, TPR, and MATH. Collinearity analysis of poplar <i>BTB</i> genes with homologs in six other species elucidated evolutionary relationships and functional conservations. RNA-seq analysis of five tissues of poplar identified <i>BTB</i> genes as playing a pivotal role during developmental processes. Comprehensive RT-qPCR analysis of 11 <i>BTB</i> genes across leaves, roots, and xylem tissues revealed their responsive expression patterns under diverse hormonal and biotic/abiotic stress conditions, with varying degrees of regulation observed in the results. This study marks the first in-depth exploration of the <i>BTB</i> gene family in poplar, providing insights into the potential roles of <i>BTB</i> genes in hormonal regulation and response to stress.