WRKY proteins, as one of the most important transcription factor families in plant, play important roles involved in many biological processes. However, studies of WRKY proteins in Gossypium barbadense remain scare. By deploying bioinformatic methods to perform a genome-wide characterization in G. barbadense genome, 180 WRKY transcription factors were identified. According to their structural features and topology of the phylogenetic tree, the GbWRKY genes were classified into three major groups (I-III). In addition, group II is further classified into five subgroups (IIa-IIe). The GbWRKY genes distributed unevenly on the 26 chromosomes. Analysis of gene duplication events demonstrated that the expansion of GbWRKY genes is mainly due to whole genome duplication and segmental duplication events. 61 GbWRKY genes showed significantly differential expression under Verticillium dahliae infection, implying that they might play important roles in interaction to V. dahlia infection. Therefore, this study provided the structure, evolution and expression pattern of GbWRKY genes, which will help further characterizing the functions of GbWRKY genes in Gossypium barbadense.