The Barn owl, Tyto alba (Scopoli, 1769), occurs worldwide and shows a considerable amount of morphological and geographical variations, leading to the recognition of many subspecies throughout the world. Yet, no comprehensive study has not been done on this species. Data from mitochondrial gene (16S Ribosomal RNA (16S)) with 569 bp length were analyzed for 41 individuals around the world. Maximum likelihood (ML), maximum parsimony (MP) and Bayesian analysis showed two distinct clades including alba clad (old world) and furcata clad (new world). The amount of genetic variation within each of these clades ranged from 0.5-1.7 but variation between clades was 3.7. This data may suggest that Barn owls of the Old World may be a separate species from those of the New World.