The western pupfish clade (Cyprinodontidae: Cyprinodon) consists of nine species that occur primarily in isolation from one another in the large area extending from the Guzmán Basin, northwestern Chihuahua, Mexico, to the Death Valley region of southeastern California and southwestern Nevada. This paper presents a reassessment of estimated divergence times based on a compilation of previously published mitochondrial DNA sequences (ND2, cytb, and control region). The results agree with previous estimates, which state that the western pupfish clade originated in the late Miocene or early Pliocene and that Cyprinodon was present in the Death Valley region by at least the middle Pliocene and possibly earlier. Enigmatically, there is little geologic evidence of late Neogene surface-water connections among the Guzmán, Lower Colorado River, and Death Valley regions. This indicates that either our knowledge of such connections is incomplete or pupfish dispersal across basin divides via small, relatively transient, surface-water connections has been more common than expected based on the low-gradient, valley-floor habitats generally occupied by this group.