The Barred Owl (Strix varia) is a common nonmigratory owl distributed across southern Canada, south to California in the west, and to Texas and Florida in the east, with isolated populations in central Mexico. We examined the genetic structure of Barred Owl populations throughout their range using 500–600 base pairs each of one nuclear and three mitochondrial genes. In 75% of the shortest trees and 64% of the bootstrapped trees, Barred Owls were not monophyletic; rather, S. v. sartorii of Mexico was separated from the remaining taxa of S. varia by S. fulvescens of Central America. Consequently, the Barred Owls of Mexico are a species-level taxon. There was a large component (32%) of genetic variance distributed among population samples from the United States and Canada because of the occurrence of two clades of haplotypes (4.8% sequence divergence) with differing geographic distributions. One clade was predominant along the Atlantic Coast and the second in the south-central United States. The two clades co-occurred from the central Gulf Coast to the Upper Midwest, and across Canada to the Pacific. Nucleotide diversity was greatest where the clades overlapped in occurrence; mismatch distributions possessed the signatures of population expansion from the southern and eastern states to northern and western locations. These results suggest two Pleistocene refugia for northern populations of Barred Owls. Diversity within populations and divergence between haplotype clades varied by an order of magnitude among the three mitochondrial genes, but each recovered the overall phylogeographic pattern. The nuclear sequences showed much less variation and differentiation.