| Summary: | The research of functional genes in <i>Acacia melanoxylon</i>, a precious and fast-growing timber species with wide adaptability, has been greatly limited due to the absence of reliable and suitable reference genes. To fill this gap, five different algorithms (comparative ΔCt, NormFinder, geNorm, BestKeeper, and RankAggreg) were employed to assess the expression stability of ten candidate genes under nine different experimental sets and their three combined groups. The results showed that <i>PP2a</i> and <i>RPL4</i> maintained stable expression in all 144 samples and a group of different tissues or organs. <i>PAT10</i> and <i>TIP41</i> were the best-performing genes in different clonal varieties, pinnate compound leaves at different growth states, salt, and indole acetic acid sets. <i>PP2a</i> and <i>PAT10</i> were the top two choices for gibberellin and abiotic stress groups. <i>PP2a</i> and <i>UBI11</i> exhibited stable expression in drought treatment. <i>UBI3</i> combined with <i>OTUD6B</i>, <i>RPL4</i>, or <i>PP2a</i> were identified as the optimal reference genes in the heat, ethephon, or exogenous hormone groups, respectively. The reliability of the selected reference genes was further confirmed by evaluating the expression patterns of <i>AmWRKY6</i> and <i>AmWRKY33</i> genes. This study provides the first comprehensive evaluation of reference gene stability in <i>A. melanoxylon</i> and promotes future research on the gene expression analysis of the species.
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