Aphid species (Hemiptera: Aphididae) are among the most serious pests for citrus cultivation throughout the world causing substantial crop damages. Accurate identification of aphids to the species level can be difficult, though being crucial for their effective management. In this study, a molecular diagnostic assay for distinguishing eleven aphid species was developed. A fragment of the mitochondrial Cytochrome Oxidase I (mtCOI) gene was used and a Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR–FLP) analysis with five restriction enzymes, based on DNA sequence polymorphisms, was applied to differentiate the eleven aphid species. This molecular technique allows aphid species at any life stage to be discriminated accurately and simply and can be a useful tool for monitoring the populations of economically important aphid species.