Aim: The islands of the south‐western Indian Ocean region are home to many endemic bird species, with their closest relatives occurring in Africa and Madagascar, Eurasia, the Sunda Islands, and the Australasian region. Among owls, the extant endemic scops owls (genus Otus) from Madagascar, Comoros, Seychelles, and Socotra are related to the Southeast Asian species, O. sunia, the Oriental scops owl. Three owl species, presumably Otus derivatives, twice the size of standard scops owls and now extinct, once inhabited the Mascarene Islands, and have been placed in a separate genus, Mascarenotus. Insular apomorphies have made their precise relationships difficult to determine. Here we investigate the phylogenetic position of these enigmatic owls. Location: The Mascarene Islands (Réunion, Mauritius, Rodrigues) in the south‐western Indian Ocean. Methods: Phylogenetic relationships were reconstructed using ancient DNA extracted from subfossil remains. Fragments of cytochrome b gene were amplified and sequenced. The ancient sequences were analysed with modern sequences of 19 ingroup Otus taxa using Bayesian and Maximum Likelihood methods. Results: The Mauritian extinct species M. sauzieri was reconstructed as the sister to both O. pauliani (Grand Comoro) and O. rutilus (Madagascar). The Rodrigues extinct species M. murivorus was the sister, in a star‐like differentiation, to the preceding clade as well as the remaining Comorian species and a clade formed by O. insularis (Seychelles) and O. sunia. Main conclusions: The ancestor of O. sunia simultaneously colonized Rodrigues Island (evolving into Otus murivorus), Madagascar, and part of the Comoros Islands around 3 million years ago. Later, presumably from Madagascar, new lineages colonized Grand Comoro and Mauritius (O. sauzieri). Independently, a more recent O. sunia ancestor colonized the Seychelles Islands and Socotra. These colonizations were probably favoured by Pliocene cyclonic events, stronger and more frequent than today. Several features, including giantism, wing reduction, and a relative decrease in skull and orbit size evolved convergently in the polyphyletic species O. sauzieri and O. murivorus.