A recent taxonomic revision of the Malagasy Scops-Owl (Otus rutilus) recognized two distinct endemic species on the island based on plumage, vocal, and morphological characters: O. rutilus (sensu stricto) from eastern humid forest formations and O. madagascariensis from western dry forest areas. An evaluation of these characters calls into question their validity for taxonomic studies, as they may be ecologically linked. To independently assess the two-species hypothesis, we used sequence data from 1449 base pairs (bp) of mitochondrial DNA (mtDNA) from 34 scops-owls obtained across the range of these two putative species. Nineteen haplotypes were detected, four of which were shared by more than one individual. Maximum sequence divergence was 0.6% (mean = 0.24%). While the most common haplotype was shared by 10 individuals originating from different eastern and western localities, 12 haplotypes were exclusive to O. rutilus and five to O. madagascariensis. An analysis of molecular variance showed significant partitioning of the genetic variability between O. rutilus and O. madagascariensis. The estimate of the divergence time between populations associated with the names O. rutilus and O. madagascariensis was 8070 years BP. Based on haplotype frequencies and sequence divergence, we conclude that there are two populations of Otus on Madagascar that started to diverge in recent geological time following an ecological parapatric model, perhaps associated with Quaternary climatic shifts. Using these results, it is inappropriate to recognize two species of Otus on Madagascar.