The California Floristic Province harbours more endemic plant and animal taxa and more identifiable subspecies than any other area of comparable size in North America. We present evidence that physical historical processes have resulted in congruent patterns of genetic diversity over the past 2–10 million years. Using a molecular clock approach we show that diversification and establishment of spatial genetic structure across six taxonomic groups coincide with the putative age of California's mountain ranges and aridification in the region. Our results demonstrate the importance of geographical barriers and climatological events to species diversification and the overall geographical structure of biodiversity. These results should facilitate conservation efforts in this biodiversity hotspot for taxa whose population genetic structure is still unknown and may suggest the potential utility of this approach in regional conservation planning efforts.