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Background:Cotton fibers are single-celled extensions of the seed epidermis, a model tissue for studying cytoskeleton. Tubulin genes play a critical role in synthesizing the microtubules (MT) as a core element of the cytoskeleton. However, there is a lack of studies concerning the systematic characterization of the tubulin gene family in cotton. Therefore, the identification and portrayal of G. hirsutum tubulin genes can provide key targets for molecular manipulation in cotton breeding. Result:In this study, we investigated all tubulin genes from different plant species and identified 98 tubulin genes in G. hirsutum. Phylogenetic analysis showed that tubulin family genes were classified into three subfamilies. The protein motifs and gene structure ofα-,β-tubulin genes are more conserved compared withγ-tubulin genes. Most tubulin genes are located at the proximate ends of the chromosomes. Spatiotemporal expression pattern by transcriptome and qRT-PCR analysis revealed that 12α-tubulin and 7β-tubulin genes are specifically expressed during different fiber development stages. However, Gh.A03G027200, Gh.D03G169300, and Gh.A11G258900 had differential expression patterns at distinct stages of fiber development in varieties J02508 and ZRI015. Conclusion:In this study, the evolutionary analysis showed that the tubulin genes were divided into three clades. The genetic structures and molecular functions were highly conserved in different plants. Three candidate genes, Gh.A03G027200, Gh.D03G169300, and Gh.A11G258900 may play a key role during fiber development complementing fiber length and strength.