As traditional Chinese medicine and edible insects, Vespa hornets have high economic value and huge market demand. Hornet breeding has become an important way to increase the income of mountain farmers in China, but the limitation of traditional identification methods has become the bottleneck of the modern development of hornet resources. In this study, a simple and reliable method for the molecular identification of typical hornets from Yunnan was successfully established. In the NCBI database, DNA sequences of hornet samples were aligned as Vespa analis, Vespa basalis, Vespa velutina, Vespa mandarinia, Vespa magnifica, Vespa binghami, Vespa soror by BLAST search. The efficiency of the PCR amplification of mitochondrial cytochrome C oxidase I (COI) gene sequences in all samples was 100% by using the optimized program. Moreover, a reference database for DNA barcodes of Vespa materials in Yunnan was constructed. This study provided a successful example for the identification of local insects of Yunnan and will be helpful to the development of wasp resources.