<p indent=0mm>Single guide RNA (sgRNA) is one of the important elements of the CRISPR/Cas9 genome editing technology system. However, studies have shown that many sgRNAs cannot work effectively. It is worth screening to verify the effectiveness of multiple design candidate sgRNAs. Instantaneous transformation of protoplasts or leaves with complete editing vectors were used to verification of the effectiveness of sgRNA in the early stage. These methods are time-consuming and laborious, and the success rate is not high, especially for cotton with low efficiency of the protoplasmic system. In this study, target sequences were designed for <italic>GhMAPKKK2</italic> and <italic>GhA</italic>E genes, and two vectors of <italic>Gh</italic>U6-5P::<italic>MAPKKK2</italic>-sgRNA-1300, <italic>Gh</italic>U6-5P::<italic>AE</italic>-sgRNA-1300 which transcibed only sgRNA were constructed and injected YZ-1 Cas9 transgenic cotton plant leaves through <italic>Agrobacterium</italic>; meanwhile, two corresponding complete CRISPR/Cas9 genome editing vectors of <italic>Gh</italic>U6-5P::<italic>MAPKKK2</italic>-sgRNA-Cas9 and <italic>Gh</italic>U6-5P::<italic>AE</italic>-sgRNA-Cas9 were constructed and injected YZ-1 wild-type cotton leaves with <italic>Agrobacterium</italic>. In addition, target sequences were designed for <italic>GhPDS</italic>,<italic> GhCLA1</italic>,<italic> GhMAPKKK2</italic>, and <italic>GhAE</italic> genes, respectively, and <italic>Gh</italic>U6-5P-2::<italic>PDS</italic>-sgRNA- CLCrVA, <italic>Gh</italic>U6-5P-2::<italic>CLA1</italic>-sgRNA-CLCrVA, <italic>Gh</italic>U6-5P-2::<italic>MAPKKK2</italic>-sgRNA-CLCrVA and <italic>Gh</italic>U6-5P-2::<italic>AE</italic>-sgRNA-CLCrVA virus delivery vectors were constructed and injected YZ-1 Cas9 transgenic cotton plant leaves through <italic>Agrobacterium</italic>. In the above experiments, the plants transformed with the empty vector were used as controls. The genomic DNA of the transformed cotton leaves was subjected to PCR and enzyme digestion, and the PCR products which were not completely digested were cloned and sequenced. The results showed that no mutation in target gene was detected in the cotton plants transformed with the <italic>Gh</italic>U6-5P::<italic>AE</italic>-sgRNA-1300, <italic>Gh</italic>U6-5P::<italic>MAPKKK2</italic>-sgRNA-Cas9 and <italic>Gh</italic>U6-5P::<italic>AE</italic>-sgRNA-Cas9, and the target genes mutation in the Cas9 transgenic plants transformed with <italic>Gh</italic>U6-5P::<italic>MAPKKK2</italic>-sgRNA-1300, <italic>Gh</italic>U6-5P-2::<italic>PDS</italic>-sgRNA-CLCrVA, <italic>Gh</italic>U6-5P-2::<italic>CLA1</italic>-sgRNA-CLCrVA, <italic>Gh</italic>U6-5P-2::<italic>MAPKKK2</italic>-sgRNA-CLCrVA and <italic>Gh</italic>U6-5P-2::<italic>AE</italic>-sgRNA-CLCrVA vector was uncovered. The types of mutations included base substitution, base deletion and base insertion. The results indicated that the strategy of using Cas9 transgenic plants as transformation recipients can efficiently and truly verify the effectiveness of sgRNA, which eliminated false negative results due to low transformation efficiency, and the strategy of using virus as vectors to deliver sgRNA was more efficient and accurate. The establishment of this sgRNA high-efficiency verification system provides an important technical basis for cotton functional genomics research.