Plasma membrane NADPH oxidases, also named respiratory burst oxidase homologues (Rbohs), play pivotal roles in many aspects of growth and development, as well as in responses to hormone signalings and various biotic and abiotic stresses. Although Rbohs family members have been identified in several plants, little is known about Rbohs in Gossypium. In this report, we characterized 13, 13, 26 and 19 Rbohs in G. arboretum, G. raimondii, G. hirsutum and G. barbadense, respectively. These Rbohs were conservative in physical properties, structures of genes and motifs. The expansion and evolution of the Rbohs dominantly depended on segmental duplication, and were under the purifying selection. Transcription analyses showed that GhRbohs were expressed in various tissues, and most GhRbohs were highly expressed in flowers. Moreover, different GhRbohs had very diverse expression patterns in response to ABA, high salinity, osmotic stress and heat stress. Some GhRbohs were preferentially and specifically expressed during ovule growth and fiber formation. These results suggest that GhRbohs may serve highly differential roles in mediating ABA signaling, in acclimation to environmental stimuli, and in fiber growth and development. Our findings are valuable for further elucidating the functions and regulation mechanisms of the Rbohs in adaptation to diverse stresses, and in growth and development in Gossypium.