Here, we provide evidence for a new aphid species from the genus Rhopalosiphum Koch, based upon an Australian survey of variation in the mitochondrial cytochrome oxidase I gene, and subsequently validated by cytochrome b, nuclear microsatellites, nuclear sequence characterized amplified region locus, and karyotypic analyses. Despite the new species being morphologically very similar to Rhopalosiphum padi (L.), there was significant genetic differentiation, with the new species being closer to the Rhopalosiphum insertum (Walker) group. Karyotypic analyses indicated a putative hybrid origin for the new species, but mitochondrial and nuclear DNA sequence data showed that the available Australian and overseas populations of Rhopalosiphum spp. did not serve as parental lineages. Diagnostic methods were developed that enabled the identification of the new species based on morphometric data and a polymerase chain reaction-restriction fragment length polymorphism based molecular technique.