Eight subspecies have been proposed within the Tawny Owl (Strix aluco) species. However, recent molecular data have challenged this view, encouraging further work in this species complex. Here we reevaluated the taxonomic status between the North-Western African Tawny Owl, S. a. mauritanica, and its closest Iberian Tawny Owl population (from the S. a. sylvatica to S. a. aluco clade) separated by the Strait of Gibraltar. The Tawny Owl is a non-migratory and territorial species, and juvenile dispersal is restricted to a few kilometers around the natal site. This limited dispersal and the barrier imposed by the Strait of Gibraltar predicted a strong differentiation between the two populations. We tested this using DNA barcoding, Bayesian phylogenetic and species delimitation analysis. We found that an 81.1% of variation is due to the intergroups variation. In addition, the inter–intraspecific distances distribution revealed a barcoding gap among the two subspecies. Also, posterior probabilities and the PAB value allowed to reject the hypothesis that observed degree of distinctiveness is due to random coalescence processes. These findings clearly support the Strait of Gibraltar as an isolating barrier for this species. The subspecific status is confirmed and species status is even suggested for S. a. mauritanica.