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Acid invertase (AINV) is a kind of sucrose hydrolase with an important role in plants.Currently, the AINV genes have not been systematically studied in cotton. In this study, a total of 92 AINV genes were identified in five cotton species. The phylogenetic analysis revealed that the AINV proteins were divided into two subgroups in cotton: vacuolar invertase (VINV) and cell wall invertase (CWINV). The analysis of gene structures, conserved motifs, and three-dimensional protein structures suggested that GhAINVs were significantly conserved. The synteny analysis showed that whole-genome duplication was the main force promoting the expansion of the AINV gene family. The cis-element, transcriptome, and quantitative real time-polymerase chain reaction (qRT-PCR) showed that some GhAINVs were possibly associated with stress response.GhCWINV4, highly expressed in PEG treatment, was cloned, and subsequent virusinduced gene silencing assay confirmed that this gene was involved in the drought stress response. Overall, this study might be helpful for further analyzing the biological function of AINVs and provide clues for improving the resistance of cotton to stress.