Microtus pennsylvanicus is represented in Mexico only by the Chihuahuan meadow vole (M. p. chihuahuensis), known from only 1 disjunct population in a small and isolated marsh in the arid lands of northern Chihuahua. Livetrapping conducted between 2000 and 2004 provided no specimens of M. p. chihuahuensis, nor was any sign of this vole observed. By the end of this study the marsh providing water had been drained, thereby destroying the vole's habitat. Surveys of other marshes in northern Chihuahua also failed to produce evidence of the species. We therefore conclude that M. p. chihuahuensis has been extirpated from its only known locality. Using “ancient” DNA from museum specimens we evaluated genetic divergence between museum specimens of M. p. chihuahuensis and 46 extant Microtus species and subspecies. Our results support the subspecific status of M. p. chihuahuensis. The loss of this subspecies is an example of population extinction, a very severe form of biodiversity loss. Until recently such losses have been mostly neglected.