The Barn Owl Tyto alba (Scopoli, 1769), occurs worldwide and shows a considerable amount of morphological and geographical variation, leading to the recognition of many subspecies around the world. But comprehensive study about this species yet to be done. In this study data from two mitochondrial genes, CYTB (620bp) and COX1 (660bp) were analyzed. Samples were collected from five different geographical areas, Palearctic, Afrotropical, Oriental, Australia and nearctic geographical region. The possibility of separation between New World and Old World species of Barn Owl was studied. Maximum likelihood (ML), Maximum parsimony (MP), Bayesian analysis and haplotype network were showed considerable genetic variation between alba clade (old world) and furcata clade (new world). This data suggested that Barn Owls of the Old World may be a separate species from those of the New World. This data showed no support for the recognition of Caribbean owl T. (f.) bargei, as a separate species. According to genetic divergence, it’s possible to introduced T. a. stertens as a separate species as well.

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