During glacial (pluvial) climatic periods, Death Valley is hypothesized to have episodically been the terminus for the Amargosa, Owens, and Mojave Rivers. Geological and biological studies have tended to support this hypothesis and a hydrological link that included the Colorado River, allowing dispersal of pupfish throughout southeastern California and western Nevada. Recent mitochondrial deoxyribonucleic acid (mtDNA) studies show a common pupfish (Cyprinodontidae) ancestry in this region with divergence beginning 3–2 Ma. We present tephrochronologic and paleomagnetic data in the context of testing the paleohydrologic connections with respect to the common collection point of the Amargosa, Owens, and Mojave Rivers in Death Valley during successive time periods: (1) the late Pliocene to early Pleistocene (3–2 Ma), (2) early to middle Pleistocene (1.2–0.5 Ma), and (3) middle to late Pleistocene (<0.7–0.03 Ma; paleolakes Manly and Mojave). Using the 3.35 Ma Zabriskie Wash tuff and 3.28 Ma Nomlaki Tuff Member of the Tuscan and Tehama Formations, which are prominent marker beds in the region, we conclude that at 3–2 Ma, a narrow lake occupied the ancient Furnace Creek Basin and that Death Valley was not hydrologically connected with the Amargosa or Mojave Rivers. A paucity of data for Panamint Valley does not allow us to evaluate an Owens River connection to Death Valley ca. 3–2 Ma. Studies by others have shown that Death Valley was not hydrologically linked to the Amargosa, Owens, or Mojave Rivers from 1.2 to 0.5 Ma. We found no evidence that Lake Manly flooded back up the Mojave River to pluvial Lake Mojave between 0.18 and 0.12 Ma, although surface water flowed from the Amargosa and Owens Rivers to Death Valley at this time. There is also no evidence for a connection of the Owens, Amargosa, or Mojave Rivers to the Colorado River in the last 3–2 m.y. Therefore, the hypothesis that pupfish dispersed or were isolated in basins throughout southeastern California and western Nevada by such a connection is not supported. Beyond the biologically predicted time frame, however, sparse and disputed data suggest that a fluvial system connected Panamint (Owens River), Death, and Amargosa Valleys, which could account for the dispersal and isolation before 3 Ma.