Plant-specific NAC transcription factors comprise a large family with diverse roles in plant development and stress regulation. In this study, 73 NAC genes from cotton (Gossypium hirsutum) EST database were identified by bioinformatic approach. Analysis of conserved amino acid residues and phylogeny reconstruction using the NAC conserved domain suggested that the Arabidopsis classification into four major groups is applicable to cotton NAC family. Among them, seven NAC genes, named as GhNAC7–GhNAC13, were characterized to encode NAC proteins that share high similarity with those plant abiotic stress-related NACs. Quantitative RT-PCR analysis indicated that the seven GhNAC genes were preferentially expressed in roots, and regulated in cotton plants under cold, abscisic acid, drought and/or high salt conditions. Our results in this comprehensive study of cotton NAC gene family provide valuable information for further exploring the roles of the NAC genes in cotton development and in response to abiotic stress.