AOC is a key enzyme in jasmonic acid (JA) biosynthesis and plays important roles in plant development and stress responses. However, the evolutionary characteristics and functional diversification of the AOC gene family in cotton remain poorly understood. In this study, AOC genes were identified and systematically characterized in four representative cotton species. Phylogenetic analysis revealed that cotton AOCs were mainly distributed in Clade II and Clade IV. Structural and promoter analyses indicated that cotton AOCs possess conserved Allene_ox_cyc domains but exhibit diversification in gene structures and regulatory elements. Synteny analysis revealed that tandem duplication and WGD/segmental duplication were the major driving forces underlying AOC gene family expansion in diploid and tetraploid cotton species, respectively. Expression analysis of GhAOC genes showed that GhAOC6 and its homologous gene GhAOC4 exhibited strong responses to Verticillium dahliae infection. Co-silencing of GhAOC4/6 significantly reduced cotton resistance to Verticillium wilt. Overall, this study provides new insights into the evolutionary conservation and functional diversification of the AOC gene family in cotton and identifies the highly homologous GhAOC4/6 pair as a candidate component associated with cotton resistance to Verticillium wilt.