Abstract: Eucalyptus cloeziana, known as 'Australian rosewood', is a valuable timber species widely cultivated in southern China. Its rapid growth and high-quality wood make it a key species for national timber reserves. This study aimed to reveal the dynamic changes of endogenous hormones during the proliferation culture of E. cloeziana tissue culture seedlings, in order to provide insights for optimizing clone-specific proliferation systems. Tissue culture seedlings of three E. cloeziana clones (75-23, YD-9, N1) were used as materials. The contents of IAA, ABA, CTK, and GA₃ in four parts (cluster buds, leaves, upper stem segments, and lower stem segments) were determined by enzyme-linked immunosorbent assay (ELISA). Statistical analyses, including ANOVA and Duncan's multiple comparisons, were employed to assess distribution characteristics, dynamic changes, and regulatory effects of hormone ratios. The results showed that: (1) Hormone distribution within the same clone exhibited tissue specificity: GA₃ content was the highest (1000-1500 pg/g) with no significant differences among parts, while CTK content was the lowest (100-250 pg/g) and remained stable. The high-content areas of IAA and ABA varied by clone; for example, in clone 75-23, IAA and ABA were significantly enriched in the upper stem segments, whereas in YD-9 and N1, IAA distribution was uniform, and ABA was significantly higher in stem segments than in leaves and cluster buds. (2) Significant differences in hormone content and composition were observed among clones: the total endogenous hormone content ranked as N1 > YD-9 > 75-23, and the hormone composition for all clones was GA₃ > ABA > IAA > CTK. (3) During proliferation, hormone contents and ratios changed significantly: as cluster buds developed into stem segments and leaves, hormone contents overall increased, with clone N1 exhibiting the largest increases in IAA (55.8%) and ABA (85.7%). The IAA/CTK ratio increased (e.g., from 2.5 to 4.5 in N1), while the GA₃/ABA ratio decreased (e.g., from 7.2 to 5.8 in N1), and clonal differences were mainly evident in the late differentiation stage. This study systematically elucidates, for the first time, the distribution and dynamics of endogenous hormones in the proliferation of E. cloeziana tissue culture seedlings, offering a theoretical basis for developing clone-specific proliferation protocols and enhancing seedling efficiency.