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The area now occupied by the Great Basin, western USA, contained paleo-fluvial systems that predated the modern-day endorheic (closed) basins. The areal extent of these paleo-fluvial systems within the southwestern Great Basin is known mainly from isolated remnants preserved in the modern mountain ranges. We document the age, extent, and tectonic disruption of Mio-Pliocene fluvial systems of the southwestern Great Basin. Synthesis of new field observations, geochemistry, and geochronology with existing studies defines two latest Miocene to Pliocene east-southeast flowing drainages that predated the modern endorheic basins. The drainage network was ultimately fragmented in Pliocene time (ca. 3.5-4 Ma). Fragmentation of the drainage network led to lake formation, drying of lakes, and the formation of isolated springs. The rapid environmental changes initiated by faulting and volcanism isolated previously interbreeding populations of spring-dwelling taxa and have caused divergent evolution since Pliocene time. Modern endemism within the region’s springs is thus a direct consequence of intraplate tectonism.