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The NAC transcription factors (NACs) are among the largest plant-specific gene regulators and play essential roles in the transcriptional regulation of both biotic and abiotic stress responses. <i>Verticillium wilt</i> of cotton caused by <i>Verticillium dahliae</i> (<i>V. dahliae</i>) is a destructive soil-borne disease that severely decreases cotton yield and quality. Although <i>NACs</i> constitute a large family in upland cotton (<i>G. hirsutum</i> L.), there is little systematic investigation of the <i>NACs’</i> responsive to <i>V.</i> <i>dahliae</i> that has been reported. To further explore the key NACs in response to <i>V. dahliae</i> resistance and obtain a better comprehension of the molecular basis of the <i>V. dahliae</i> stress response in cotton, a genome-wide survey was performed in this study. To investigate the roles of <i>GhNACs</i> under <i>V.</i> <i>dahliae</i> induction in upland cotton, mRNA libraries were constructed from mocked and infected roots of upland cotton cultivars with the <i>V. dahliae</i>-sensitive cultivar “Jimian 11” (J11) and <i>V. dahliae</i>-tolerant cultivar “Zhongzhimian 2” (Z2). A total of 271 <i>GhNACs</i> were identified. Genome analysis showed <i>GhNACs</i> phylogenetically classified into 12 subfamilies and distributed across 26 chromosomes and 20 scaffolds. A comparative transcriptome analysis revealed 54 <i>GhNACs</i> were differentially expressed under <i>V.</i> <i>dahliae</i> stress, suggesting a potential role of these <i>GhNACs</i> in disease response. Additionally, one <i>NAC090</i> homolog, <i>GhNAC204</i>, could be a positive regulator of cotton resistance to <i>V. dahliae</i> infection. These results give insight into the GhNAC gene family, identify GhNAC<i>s’</i> responsiveness to <i>V.</i> <i>dahliae</i> infection, and provide potential molecular targets for future studies for improving <i>V. dahliae</i> resistance in cotton.