Climate change during the late Quaternary has been implicated as the cause of both massive range shifts and extinction events. We combined molecular marker data and previously published fossil data to reconstruct the late Quaternary history of a grassland-dependent species, the black-footed ferret (Mustela nigripes), and to determine whether populations from Pleistocene refugia in the Columbia Basin, eastern Beringia, and Great Plains persisted into the Holocene and Recent eras. Using DNA extracted from 97 museum specimens of extirpated populations, we amplified 309 bp of the mtDNA control region, and 8 microsatellite markers from the nuclear genome. Overall haplotype diversity from 309 base pairs (bp) of mitochondrial DNA (mtDNA) control region was low (5 haplotypes, nucleotide diversity = 0.001 ± 0.001 SD) and was contained within a single phylogenetic clade. The star phylogeny and unimodal mismatch distribution indicated that a rapid range expansion from a single Pleistocene refugium occurred. Microsatellite data corroborated this genetic pattern: populations from the mixed grasslands of the Great Plains had significantly higher expected heterozygosity and allelic richness than populations to the west (H_E = 0.66 versus 0.41, AR = 4.3 versus 2.7, respectively), and 𝜰, a measure of relative population size, was substantially greater in the east than west (2.4 versus 0.7). We infer from these data that black-footed ferrets rapidly colonized western ecoregions in a stepwise fashion from the Great Plains to the intermountain regions of the Rocky Mountains and the Colorado Plateau after the last ice age. It appears that glacial retreat and global warming caused both range expansion and localized extinction in this North American mustelid species.