Abstract: 【Objective】 To explore the role of GHWAT1-35 gene in the development of cotton fibers. 【Methods】 In this study, the fiber at different developmental stages post-anthesis of Gossypium hirsutum variety Xi9 were used as materials.The full-length cDNA sequence of the GHWAT1-35 gene was successfully cloned and subjected to bioinformatics analysis, real-time fluorescence quantitative (qRT-PCR) analysis, and subcellular localization. 【Results】 The length of the GHWAT1-35 gene was 1125 bp, encoding 374 amino acids, with a relative molecular weight is 40.23 kDa and a theoretical isoelectric point of 8.74.Protein multiple sequence alignment and construction of a systematic evolutionary tree analysis showed that the GHWAT1-35 protein was most closely related to the Gossypium arboreum.The expression of the GHWAT1-35 gene significantly increased at 15 days after flowering, and subcellular localization predicted that the protein was located on the cytoplasmic membrane.The GHWAT1-35 gene was recombined with the pCAMIA1300-35S-YFP vector to construct a fusion expression vector, which was then transformed into Agrobacterium using the freeze-thaw method and after being injected into tobacco.The GHWAT1-35 protein was observed to be located on the cytoplasmic membrane. 【Conclusion】 The GHWAT1-35 gene plays an important role in fiber development, providing a foundation for further exploration of its biological function in Gossypium hirsutum.