Hyperhydricity is a frequently occurring physiological disorder in plant tissue culture that impairs the regeneration and survival of vitrified plant materials, leading to significant losses and complicating research applications. Although numerous studies have focused on strategies to mitigate hyperhydricity, its molecular mechanisms remain poorly characterized. In this study, transcriptome sequencing and endogenous hormone content analysis were conducted on hyperhydric and normal callus of flax (Linum usitatissimum L.). Transcriptomic analysis revealed 2698 differentially expressed genes (DEGs) between these two tissue types. Pathway analysis through KEGG demonstrated that these DEGs predominantly linked to metabolic processes including phenylpropanoid formation, MAPK signaling cascades, and plant hormone signal transduction. Moreover, quantification of endogenous hormone levels indicated a significant reduction in all hormones except cytokinins (ZRs) in hyperhydric tissues. The observed disruption in endogenous hormone levels suggests its potential role in the development of plant vitrification. These findings provide valuable insights into the molecular processes underlying hyperhydricity, contributing to a more comprehensive understanding of this phenomenon.