Objective To conduct molecular identification of the common Siphonaptera species from the Alataw pass of China-Kazakhstan border based on the mitochondrial cytochrome C oxidase subunit I (CO Ⅰ) gene. Methods In May 2017, parasitic Siphonaptera on the body surface of rodents was collected from the Alataw pass of China-Kazakhstan border. DNA was extracted after morphological identification. The mitochondrial CO Ⅰ gene was amplified by PCR and sequenced. The sequencing results were analyzed by BLAST, and the phylogenetic tree was constructed using MAGE 6.0 software. Results The morphological and molecular biological identification showed 100% consistency between Echidnophaga oschanini and Ctenocephalides felis felis. Meanwhile, the mitochondrial CO Ⅰ sequences of Xenopsylla minax and Nosopsyllus laeviceps laeviceps were reported for the first time in the world. Molecular biological identification and morphological identification of the four species of Siphonaptera were in good agreement. The four species of Siphonaptera in China-Kazakhstan border were highly similar to Siphonaptera species in other countries. Conclusion DNA barcoding technique can identify the species of Siphonaptera effectively and provide a scientific basis for rapid and accurate identification of host and control of foreign medical vectors at ports.