Recent molecular studies have proved beneficial in providing taxonomic resolution within the
Octopus vulgarisspecies 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 vulgarisspecies 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. insularisto Ascension and St Helena Islands. Octopus vulgarisand two other member species of the O. vulgarisspecies complex, O. tetricusand O. cf. tetricusformed a highly supported monophyletic clade (BS = 99, PP = 1). Interspecific distances between the O. mimusgroup (O. mimus, O. bimaculoides, O. mayaand O. insularis)and the O. vulgarisspecies group (O. vulgaris, O. tetricusand 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.