Abstract <p><i>Hydrangea arborescens</i> ‘Annabelle’ is characterized by its large and beautiful flowers and strong adaptability, making it an emerging woody ornamental plant with unique advantages in the global horticultural market. In this study, we conducted an in-depth transcriptome sequencing and analysis of four critical stages during the rooting process of ‘Annabelle’ tissue culture seedlings using RNA sequencing technology. The aim was to elucidate the molecular mechanisms of adventitious root development in <i>Hydrangea arborescens</i>. By performing GO and KEGG enrichment analyses and constructing a gene co-expression network (GCN), we identified key genes that play regulatory roles in specific stages of adventitious root development. This provides a solid foundation for theoretical research in related fields. The results showed that most of the annotated genes and transcription factors in the four stages were related to plant hormones, glycolytic metabolism, stress response, energy metabolism, photoperiod, phenylpropanoid biosynthesis, and circadian rhythm. This study provides insights into the molecular mechanisms of adventitious root development in Hydrangea and aids in the research of other closely related species.</p>