(1) Background: Brassinosteroids (BRs) are important hormones involved in almost all stages of plant growth and development, and sterol dehydrogenase is a key enzyme involved in BRs biosynthesis. However, the sterol dehydrogenase gene family of Populus yunnanensis Dode (P. yunnanensis) has not been studied. (2) Methods: The PyDET2 (DEETIOLATED2) gene family was identified and analyzed. Three genes were screened based on RNA-seq of the stem tips, and the PyDET2e was further investigated via qRT-PCR (quantitative real-time polymerase chain reaction) and subcellular localization. (3) Results: The 14 DET2 family genes in P. yunnanensis were categorized into four groups, and 10 conserved protein motifs were identified. The gene structure, chromosome distribution, collinearity, and codon preference of all PyDET2 genes in the P. yunnanensis genome were analyzed. The codon preference of this family is towards the A/U ending, which is strongly influenced by natural selection. The PyDET2e gene was expressed at a higher level in September than in July, and it was significantly expressed in stems, stem tips, and leaves. The PyDET2e protein was localized in chloroplasts. (4) Conclusions: The PyDET2e plays an important role in the rapid growth period of P. yunnanensis. This systematic analysis provides a basis for the genome-wide identification of genes related to the brassinolide biosynthesis process in P. yunnanensis, and lays a foundation for the study of the rapid growth mechanism of P. yunnanensis.

(1) Background: Brassinosteroids (BRs) are an important hormone that participates in almost all stages of plant growth and development, and sterol dehydrogenase is a key enzyme that is involved in the biosynthesis of BRs. However, the steroid dehydrogenase gene family in Populus yunnanensis Dode (P. yunnanensis) has not been studied; (2) Methods: The PyDET2 gene family was identified and bioinformatics analyzed. Three genes were screened based on the RNA-seq of stem tip, the PyDET2e was further investigated by RT-qPCR (real-time quantitative polymerase chain reaction) and subcellular localization; (3) Results: We identified 14 DET2 family genes in P. yunnanensis that were classified into 4 groups according to sequence similarity and phylogenetic relationship, and 10 conserved protein motifs had been identified. The gene structure, chromosome distribution, collinearity, and codon preference of all PyDET2 genes identified in the genome of P. yunnanensis were analyzed. The codon of this family is biased to the end of A/U, and natural selection has a great influence on it. The expression level of PyDET2e in September was higher than in July, and it was significantly expressed in stems, stem tips, and leaves. The PyDET2e protein was localized in chloroplasts; (4) Conclusions: The PyDET2e is involved in the development of P. yunnanensis in the rapid growth stage. The systematic analysis provided a basis for the genome-wide identification of genes related to brassinolide biosynthesis processes in P. yunnanensis, and laid a foundation for the study on the mechanism of rapid growth of P. yunnanensis.