A class of proteins, 1-aminocyclopropane-1-carboxylate oxidase (ACO), is required in the final step of production of ethylene from its immediate precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Despite the crucial and regulatory role of ACO gene family in the fiber development, it has not been thoroughly analyzed and annotated in G. barbadense genome. In the present study, we have identified and characterized all isoforms of ACO gene family from genomes of Gossypium arboreum, G. barbadense, G. hirsutum and G. raimondii. Phylogenetic analysis classified all ACO proteins into six distinct groups on the basis of maximum likelihood. Gene locus analysis and circos plots indicated the distribution and relationship of these genes in cotton genomes. Transcriptional profiling of ACO isoforms in G. arboreum, G. barbadense and G. hirsutum fiber development exhibited the highest expression in G. barbadense during early fiber elongation. Moreover, the accumulation of ACC was found highest in developing fibers of G. barbadense in comparison with other cotton species. ACO expression and ACC accumulation correlated with the fiber length in cotton species. Addition of ACC to the ovule cultures of G. barbadense significantly increased fiber elongation while ethylene inhibitors hindered fiber elongation. These findings will be helpful in dissecting the role of ACOs in cotton fiber development and pave a way towards genetic manipulations for fiber quality improvement.