Recent molecular studies have proved beneficial in providing taxonomic resolution within the Octopus vulgaris species complex, therefore aiding in the appropriate management of this high value global fisheries resource. This study used the mitochondrial ‘barcode of life’ gene Cytochrome Oxidase subunit I (COI) to investigate the identity of shallow-water benthic octopuses in the mid-Atlantic Ocean and their relationship to members of the Octopus vulgaris species complex. Maximum likelihood and Bayesian phylogenetic inference placed individuals collected from two tropical islands, Ascension and St Helena, into a highly supported monophyletic clade with the North Brazilian species O. insularis (BS = 81, PP = 1), extending the known distribution of O. insularis to Ascension and St Helena Islands. Octopus vulgaris and two other member species of the O. vulgaris species complex, O. tetricus and O. cf. tetricus formed a highly supported monophyletic clade (BS = 99, PP = 1). Interspecific distances between the O. mimus group (O. mimus, O. bimaculoides, O. maya and O. insularis) and the O. vulgaris species group (O. vulgaris, O. tetricus and O. cf. tetricus) ranged from 14.7–26.0%, and an estimated date of divergence suggests these groups diverged from a common ancestor between 19.0 and 40.9 million years ago.