A primary goal of conservation genetics is the discovery, delimitation and protection of phylogenetic lineages within sensitive or endangered taxa. Given the importance of lineage protection, a combination of phylogeography, historical geology and molecular clock analyses can provide an important historical context for overall species conservation. We present the results of a range-wide survey of genetic variation in the California tiger salamander, Ambystoma californiense, as well as a summary of the past several million years of inundation and isolation of the Great Central Valley and surrounding uplands that constitute its limited range. A combination of population genetic and phylogenetic analyses of mitochondrial DNA variation among 696 samples from 84 populations revealed six well-supported genetic units that are geographically discrete and characterized by nonoverlapping haplotype distributions. Populations from Santa Barbara and Sonoma Counties are particularly well differentiated and geographically isolated from all others. The remaining units in the Southern San Joaquin Valley, Central Coast Range, Central Valley and Bay Area are separated by geological features, ecological zone boundaries, or both. The geological history of the California landscape is consistent with molecular clock evidence suggesting that the Santa Barbara unit has been isolated for at least 0.74–0.92 Myr, and the Sonoma clade is equally ancient. Our work places patterns of genetic differentiation into both temporal- and landscape-level contexts, providing important insights into the conservation genetics of the California tiger salamander.