【Objectives】Chalcone isomerase (CHI), functioning as a pivotal rate-limiting enzyme in the flavonoid biosynthetic pathway, exerts a decisive regulatory influence on the biosynthesis and accumulation of plant secondary metabolites. Flavonoids, as indispensable secondary metabolites, are intricately associated with multiple vital physiological processes, including plant growth, development, and stress resilience, playing an irreplaceable role in maintaining plant homeostasis and ecological adaptability. However, despite the fast-growing tree Paulownia fortunei, which possesses substantial ecological and economic value and serve as a key species for forestry development, systematic identification and functional exploration of its CHI gene family remain unexplored. This study is devoted to conducting a comprehensive genome-wide identification of all CHI gene family members in P. fortunei. We systematically dissect their physicochemical properties, evolutionary relationships, and structural characteristics, and further clarify their expression patterns across diverse tissues and under various abiotic stress conditions. This research aims to lay a robust theoretical foundation for elucidating the biological functions of CHI genes in the secondary metabolism and stress resistance of P. fortunei, thereby providing critical scientific insights to enhance the species' adaptability and utilization value, and supporting its sustainable development in forestry.【Method】By combining bioinformatics with molecular biology techniques,at the initial stage, the PfCHI gene family was systematically pinpointed through comprehensive genome-wide screening, which was effectively leveraged by specialized bioinformatics tools, ensuring the accuracy and thoroughness of gene identification. The physicochemical attributes of the identified genes, including protein molecular weight, isoelectric point, and hydrophobicity, were precisely predicted using established computational algorithms, providing a solid foundation for understanding their molecular characteristics.To clarify evolutionary relationships, a phylogenetic tree was constructed, offering clear insights into the evolutionary trajectory of the PfCHI family. The gene structures and promoter regions were thoroughly characterized, with a focus on unraveling potential regulatory elements that may govern gene expression. To delve into the expression patterns of PfCHI genes, transcriptome data was synergized with reverse transcription quantitative polymerase chain reaction (RT-qPCR) for cross-validation, enhancing the reliability of the results. The expression levels of PfCHI genes were meticulously examined across various tissues, such as roots, stems and leaves, as well as under NaCl treatment (salt stress) and PEG6000 treatment (drought stress), aiming to reveal their tissue-specific expression characteristics and stress responsiveness.【Result】A total of five PfCHI genes were successfully identified through systematic genome-wide screening, with each gene precisely mapped to five distinct chromosomes, revealing a well-defined genomic distribution pattern. Phylogenetic analysis further categorized these PfCHI genes into two distinct subfamilies, namely type Ⅰ and type Ⅲ, which clearly reflects functional divergence shaped during evolutionary processes, providing critical insights into the evolutionary trajectory and functional specialization of the PfCHI gene family.Promoter analysis demonstrated that the upstream regulatory regions of the PfCHI genes were replete with a variety of cis-acting elements. These elements were closely associated with light response, hormone signaling pathways, and stress response, implying that the PfCHI genes have the potential to integrate multiple environmental cues and hormonal regulatory signals, thereby participating in the fine-tuning of plant physiological processes.RT-qPCR results indicated that all PfCHI genes exhibited the highest expression levels in leaves, strongly suggesting that leaves serve as the primary site for flavonoid biosynthesis in P. fortunei. Moreover, these genes were markedly induced under both salt and drought stress conditions, highlighting their crucial involvement in the plant’s abiotic stress response mechanisms.【Conclusion】This study marks the pioneering effort in conducting a systematic identification and in-depth characterization of the CHI gene family within P. fortunei. Five distinct PfCHI genes were successfully pinpointed, with their genomic distribution spanning across five chromosomes and their evolutionary classification clearly delineated into two subfamilies. This comprehensive delineation offers a detailed panorama of the family’s structural architecture and evolutionary dynamics, laying a solid foundation for subsequent functional investigations.The promoter regions of these genes are replete with stress-responsive cis-elements, and their expression patterns are markedly induced under stress conditions, collectively underscoring the genes’ pivotal role in mediating stress adaptation responses. These groundbreaking findings not only establish a fundamental theoretical framework for elucidating the flavonoid biosynthesis pathway and the intricate molecular mechanisms underlying stress resistance in P. fortunei, but also precisely identify a cluster of core candidate genes endowed with substantial application prospects.The identified genes serve as invaluable genetic resources and provide robust technical backing for molecular breeding initiatives aimed at enhancing stress resistance and optimizing secondary metabolite profiles in P. fortunei.This study charts a clear pathway for the sustainable development and utilization of this economically and ecologically significant tree species, propelling advancements in tree biology research.