The black bean aphid, Aphis fabae Scopoli is a polyphagous aphid causing significant damage to several cultivated and uncultivated plants. In the present work, we have used COI barcoding to distinguish between three subspecies of A. fabae, frequently found in Tunisia, namely A. f. fabae, A. f. solanella and A. f. cirsiiacanthoidis. Results of our study revealed that COI barcoding was efficient to generate haplotypes discriminating A. f. fabae from the two remaining subspecies. Indeed, among seven haplotypes, identified and named H1–H7, five were found exclusively in A. f. fabae, one represented exclusively A. f. cirsiiacanthoidis, and one was shared by A. f. cirsiiacanthoidis and A. f. solanella. However, the differentiation of Aphis f. fabae from A. f. solanella and A. f. cirsiiacanthoidis was not further supported by a phylogenetic distinction, as it was revealed by neighbour-joining and maximum parsimony phylogenies. Such a coherent genetic structure of the A. fabae complex, not governed by the subspecies factor, as inferred from our present mitochondrial (mt)DNA data, is in agreement with previous analyses based on genomic DNA. Therefore, we hypothesize that the phenotypical and physiological processes having led to differentiation between subspecies would have arisen from weak selection pressures, thus resulting in little genetic change between subspecies. In combination with additional still-to-come data from additional markers, results of this study will be important to constitute a groundwork for pest integrated management.