Abstract Morphologically similar species occur in various groups of insects, including aphid pests. In Europe, Aphis frangulae Kaltenbach and Aphis gossypii Glover (sometimes considered as subspecies) are differentiated usually on the basis of life cycle and host plant. We used a sexual population of A. frangulae collected on the primary host and samples of A. gossypii collected on cucurbits or cotton for the development of molecular markers. DNA sequence data for the gene encoding cytochrome b and for the barcode region of cytochrome oxidase I, as well as a length polymorphism for an intron in the sodium channel para-type gene discriminated unambiguously between the two taxa. These markers were also used as identification keys for aphids collected on crops belonging to the Solanaceae. The cytochrome b marker differentiates host-related Aphis gossypii haplotypes, and the para-type gene intron might be suitable for the resolution of taxonomic problems in other aphid species complexes.