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Our analysis of the genetic structure of northern spotted owls across most of the range of the subspecies allowed us to test for genetic discontinuities and identify landscape features that influence the subspecies’ genetic structure. Although no genetic breaks were found in northern spotted owls, landscape features were important in structuring genetic variation. Dry, low elevation valleys and the high elevation Cascade and Olympic Mountains restricted gene flow, while the lower Oregon Coast Range gene flow, acting as a “genetic corridor.” The Columbia River did not act as a barrier, owls readily fly over this large river. Thus, even in taxa such as northern spotted owls with potential for long-distance dispersal, landscape features can have an important impact on gene flow and genetic structure.