Cotton is a significant cash crop and the primary source of natural fiber globally. Among the numerous diseases encountered in cotton production, Verticillium wilt is one of the most serious, caused by the pathogen Verticillium dahliae (V. dahliae). Unfortunately, there are no effective targeted methods to combat this disease. Genomic resources for Verticillium wilt resistance primarily exist in Gossypium barbadense (G. barbadense). Regrettably, there have been limited transcriptomic comparisons between V. dahliae-resistant and -susceptible varieties of G. barbadense due to the scarcity of susceptible resources. In this study, we conducted a transcriptome analysis on both V. dahliae-resistant and -susceptible varieties of G. barbadense at the 0, 12, 24 and 48 hours after V. dahliae inoculation. This comparative transcriptome analysis yielded high-quality data and offered new insights into the molecular mechanisms underlying cotton's resistance against this destructive pathogen.