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Sequence variation was examined in the cytochrome b region of the mitochondrial genome of Burrowing Owls (Athene cunicularia) from North and South America, and compared with the Elf Owl (Micrathene whitneyi), Barred Owl (Strix varia) and Eastern Screech Owl (Otus asio). Attempts to done and sequence the control region of the mitochondrial genome resulted in sequences that appeared to be nuclear copies of that region. Cytochrome b sequences revealed a genetic split between Burrowing Owl populations from North and South America. This split may date back 2 million yr to the connection of these continents via the isthmian land bridge. Additional population structure appears to be of Pleistocene origin or more recent. Data indicate a possible North American origin for Burrowing Owls arid subsequent dispersal via the land bridge to the South American continent. The depth of the split between Burrowing Owls from North and South America is consistent with species-level distinction. Additional data from nuclear markers, morphology and/or ecological indicators, such as behavior or vocalizations, will be necessary to confirm these results.