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