Abstract: Abstract: Objective To systematically characterize the R2R3-MYB transcription factor family in Neolamarckia cadamba, analyze their expression profiles across different tissues and in response to phytohormone treatments, and thereby establish a foundation for exploring their biological functions in N. cadamba. Methods The R2R3-MYB family in N. cadamba was systematically characterized through a genome-wide bioinformatics approach, encompassing analyses of phylogeny, gene structure, physicochemical properties, chromosomal distribution, and cis-elements. Subsequently, the expression patterns of these genes were investigated using transcriptome data from various tissues and in response to hormone treatments such as salicylic acid (SA). Results A total of 148 R2R3-MYB genes were identified and mapped across all 20 chromosomes. Phylogenetic analysis classified these genes into 33 subgroups, with subgroup S15 containing the largest number of members (nine). Syntenic gene pairs were found to have close branch distances and conserved exon-intron structures. Analysis of promoter regions predicted the presence of cis-elements associated with hormone, light, and stress responses. Transcriptome profiling demonstrated that most NcMYB genes were expressed across different tissues, with NcMYB95 and NcMYB88 highly expressed in secondary xylem. Transcriptome profiling demonstrated that MYB genes exhibit widespread responsiveness to SA as well as other hormonal and abiotic cues, suggesting diverse biological functions. By integrating expression patterns with phylogenetic relationships and in silico predictions of transcription factor binding sites in cadambine-biosynthetic genes, we identified NcMYB79, NcMYB100, NcMYB73, NcMYB97, NcMYB80, and NcMYB71 as candidate regulators of cadambine biosynthesis. Conclusion This study presents a genome-wide identification of the R2R3-MYB family members in N. cadamba. Six candidate genes potentially involved in the regulation of cadambine biosynthesis were identified, laying a molecular foundation for further unraveling the transcriptional regulatory mechanisms of cadambine biosynthesis and providing important guidance for synthetic biology research on cadambine in N. cadamba..