Abstract:Here we characterize the genetic structure of Black Salamanders (Aneides flavipunctatus) in the Klamath Mountains of northwestern California and southwestern Oregon using mitochondrial and nuclear DNA sequences. We hypothesized that the Sacramento, Smith, Klamath, and Rogue River watersheds would represent distinct genetic populations based on prior ecological results, which suggest that Black Salamanders avoid high elevations such as the ridges that separate watersheds. Our mitochondrial results revealed two major lineages, one in the Sacramento River watershed, and another containing the Klamath, Smith, and Rogue River watersheds. Clustering analyses of our thirteen nuclear loci show the Sacramento watershed population to be genetically distinctive. Populations in the Klamath, Smith, and Rogue watersheds are also distinctive but not as differentiated and their boundaries do not correspond to watersheds. Our historical demographic analyses suggest that the Sacramento population has been isolated from the Klamath populations since the mid-Pleistocene, with negligible subsequent gene flow (2 Nm ≤ 0.1). The Smith and Rogue River watershed populations 658show genetic signals of recent population expansion. These results suggest that the Sacramento River and Klamath River watersheds served as Pleistocene refugia, and that the Rogue and Smith River watersheds were colonized more recently by northward range expansion from the Klamath.