Cotton is one of the most important economic crops in the world, as well as the most important fiber crop and important oil crop. Xinjiang is the largest region of cotton producing in China. However, cotton production has affected by drought stress become the main abiotic stress factor . It is an effective way to address the scarcity of cotton drought resistance resources and improve water resource utilization is exploiting and utilizing key regulatory factors for drought resistance and cultivating drought resistant cotton varieties. According to the principle of CRISPR/Cas9 technology, two 20 bp targeted sequences were designed in the first exon of the GhNAC3 genome sequence. The gene shuffling of non-specific target sites was eliminated using the blast analysis. The oligonucleotides of two target sites were inserted into the gRNA-atU6 plasmid vector, and then were amplified twice by PCR technology to construct atU6-GhNAC3 expression-boxes. The recombinant CRISPR-GhNAC3 vector was obtained by linking two expression-boxes to CRISPR/Cas9(pRGEB32-7) vector. The CRISPR-GhNAC3 vector was transformed into the cotton of YZ-1 through agrobacterium mediated method. The T0 generation of transgenic plants was obtained by detection of Cas9 protein gene , and the positive transgenic plants were selected through PCR method. The target sites of each T0 generation from were determined [收稿日期： 基金项目：新疆农垦科学院院级项目（03110004）;八师石河子市中青年科技创新骨干人才计划（2022RC03）；第一师阿拉尔市科技计划项目（2022NY10）；新疆农垦科学院创新团队 作者简介：王志军（1985-），博士，助理研究员，作物遗传育种，E-mail：wzjshihezi@163.com *通信作者：马盼盼，副研究员，研究方向，作物抗逆种质创新及育种，E-mail：mp20044681@163.com]via PCR and sequencing method, and finally the mutated genotypes were identified. After Cas9 protein gene identification, 30 positive strains were obtained, and 10 of them were selected from the positive plants for editing type sequencing analysis. It was found that all 7 strains underwent editing in the target region. The editing type was mainly base fragment deletion, with a deletion fragment size of 3-28 bp. T1 was planted in the field, and 3 mutants with both yellowing and dwarfing were obtained. The results showed that using CRISPR/Cas9 technology to edit the GhNAC3 sequence, plants with phenotypic variations were obtained, fully demonstrating that the gene is involved in the regulation of cotton growth and development.