Sorghum aphid (Melanaphis sacchari), greenbug (Schizaphis graminum) and corn leaf aphid (Rhopalosiphum maidis) are the main pests of sorghum in China. Genotype characterization and/or biotype identification of pests is the first step for cereal breeding programme to obtain resistant cultivars. DNA barcoding technique provides an effective tool for species and biotype diagnosis in various organism. In this study, we sequenced a fragment of 680 bp of cytochromecoxidase I (COI) gene from above aphid species collected from 16 sorghum growing regions in China. The results revealed that (i) No polymorphism was found in the mitochondrial DNA from different sorghum aphid and greenbug populations. The diversity in these two species was not as much as we expected, (ii) Three haplotypes were identified in corn leaf aphid clones. The most common haplotype was the same as that in Canada, USA and UK and (iii) Mitochondrial COI sequences could effectively distinguish different aphid species, and further different haplotypes within an aphid species.