Carbonic anhydrases (CAs) are critical metalloenzymes, widely exist in organisms, which involve in many physiological processes, including response to adverse environmental conditions. Although CA genes have been comprehensive identified and analyzed in numerous plants, there are a few of reports in cotton. Therefore, we conducted an exhaustive research for CA genes from two tetraploid cotton species and their ancestral species. A total of 138 CA genes were found, and 45 of them belonged to Gossypium hirsutum. Phylogenetic relationships and sequences analysis showed that CA genes were categorized into three distinct subtypes: α-type, β-type and γ-type. The exon numbers of β-type members were highly variable. Various types of cis-elements, including drought inducibility, were identified in CA genes, suggesting that CA genes might be involved in the regulation of drought stress response. qRT-PCR was applied to assess the gene expression level in various tissues under drought stress. The results indicated that the expression levels of GhαCA4-D, GhβCA1-A, GhβCA1-D, GhβCA3-D and GhβCA6-D were significantly higher in leaves than that in stems and roots. The expression of GhαCA4-A, GhαCA8-A, GhαCA4-D, GhβCA3-D, GhβCA6-D and GhγCAL1-D was significantly upregulated in roots at severe drought treatment. The functions of GhαCA4-D, GhβCA6-D and GhγCA2-D were analyzed using virus-induced gene silencing (VIGS) technology. Compared to the controls, GhγCA2-D-silenced upland cotton seedlings were more sensitive to salt stress. However, the drought tolerance of GhαCA4-D and GhβCA6-D silenced plants was significantly decreased. Stomatal density, width and area were significantly higher in TRV:GhβCA6-D compared to TRV:00 inoculated plants. GhαCA4-D silenced plants were susceptible to oxidative stress, and silencing GhαCA4-D induced leave cell death. Our results will assist to make clear the regulatory mechanism of CA genes under abiotic stress.