The geographic distribution of Octopus mimus Gould, 1852 is unclear, as most records are restricted to coastal localities of Peru and Chile, and some references have mentioned its presence off Central America. It is unknown whether this octopus is found off Ecuador as two previous unpublished records have suggested. The aim of the present study is to identify genetically the main octopus captured in a marine protected area known as Reserva de Produccion Faunistica Marino-Costera Puntilla de Santa Elena (REMACOPSE) off the Santa Elena peninsula, Ecuador. Samples collected from the local fishery were used to test the presence of this species based on the sequences of three mitochondrial markers and using a Bayesian approach. The phylogenetic analysis confirms that O. mimus inhabits the REMACOPSE. The results also indicate that the octopus specimens captured in the fishery from this marine protected area, are more closely related to O. mimus specimens from Central America than those from South America. The genetic identification of two groups of O. mimus could be associated with the different marine environmental conditions of the two biogeographic provinces. The finding in this study represents an important step for posterior research on the biology and fishery of octopus in Ecuador.