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Secondary cell wall (SCW) biosynthesis is an important stage of the cotton fibre development, and its transcriptional regulation is poorly understood. We selected the Gossypium hirsutum GDSL (GhGDSL) lipase/hydrolase gene (CotAD_74480), which is expressed during SCW biosynthesis (19 through to 25 days postanthesis; DPA), for study. T₁‐transgenic cotton lines expressing the β‐glucuronidase (gus) reporter under the control of a 1026‐bp promoter fragment of GhGDSL (P_GhGDSL) showed 19 DPA stage‐specific increase in GUS expression. 5′ deletion indicated that the 194‐bp fragment between –788 and –594 relative to the transcription start site was essential for this stage‐specific expression. Site‐directed mutagenesis of eight transcription factor binding sites within P_GhGDSL demonstrated that the MYB1AT motif (AAACCA) at –603/–598 was critical for the 19 DPA‐specific reporter gene expressions …