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This study identified 13 <i>GhTIR1</i>/<i>AFB</i> members in <i>G. hirsutum</i> through bioinformatics methods and divided them into three subgroups by phylogenetic tree analysis. Motif and gene structure analysis showed that the genes in this family were highly conserved. Promoter <i>cis</i>-acting element analysis found that the promoters of <i>GhTIR1</i>/<i>AFBs</i> contained a large number of <i>cis</i>-acting elements in response to growth and development and abiotic stress. Further RT-qPCR results showed that <i>GhTIR1</i>/<i>AFB</i> genes responded to various abiotic stresses such as IAA, ABA, cold, and heat, and the expression levels of each gene changed obviously, especially <i>Gh_D08G0763</i> (<i>GhTIR1</i>), which responded significantly to cold injury. Using VIGS (virus-induced gene silencing) technology to silence <i>Gh_D08G0763</i> in the cold-tolerant cotton variety <i>ZM36</i>, it was found that the resistance of <i>ZM36</i> to cold damage was significantly reduced. The physiological response mechanism of the <i>Gh_D08G0763</i> in resisting cold damage was further analyzed through trypan blue staining of leaves and determination of enzyme activity levels. This study provided effective genetic resources for cotton cold-tolerance breeding.