Abstract. Sequences of the mitochondrial cytochrome b gene (930 base pairs) were used to examine patterns of variation within and between Eastern (Megascops asio) and Western (M. kennicottii) Screech-Owls, and to assess taxonomic affinity of Flammulated Owls (Otus flammeolus) and Whiskered Screech-Owls (M. trichopsis). Analyses support monophyly of the New World Megascops, a sister-group relationship between O. flammeolus and New World forms of Megascops, rather than with Old World Otus, and a closer relationship between the mostly North American M. trichopsis and South American Megascops than between M. trichopsis and North American Megascops. Megascops asio and M. kennicottii formed two distinct monophyletic clades, supporting species-level designations as suggested by morphology and song. Evidence for distinctive subspecies of eastern and western forms of screech-owls was less compelling. In the M. asio group, M. a. mccallii was the only subspecies with a unique haplotype; other subspecies within M. asio were phylogenetically indistinguishable. Subspecies within M. kennicottii were partitioned into three geographic groups, and differences are probably the result of barriers to gene flow (e.g., mountains above 2300 m), which are more pronounced throughout the distribution of M. kennicottii than in the distribution of M. asio.