Species that have experienced population reduction provide valuable case studies for understanding genetic responses to demographic change. Pronghorn (Antilocapra americana) were once widespread across the North American plains but were subject to drastic population reductions due to overexploitation and habitat fragmentation during the late 19th and early 20th centuries. A. a. peninsularis and A. a. sonoriensis, 2 pronghorn subspecies that inhabit the southern edge of the species' distribution, are almost extinct and now breed almost exclusively in captivity. We therefore sequenced the complete mitochondrial control region and genotyped 18 microsatellite loci in 109 individuals to evaluate the impact of population bottlenecks, captive breeding, small population sizes, and isolation on the genetic composition of captive populations of these 2 subspecies. We found extremely low levels of genetic diversity in both subspecies. The 2 subspecies showed high and significant genetic differentiation, indicating the absence of historic and recent gene flow despite their geographic proximity within the Sonoran Desert. Historical effective population size estimates for the 2 subspecies were inferred to be similar, whereas the Sonoran pronghorn has a contemporary effective size (Ne) more than twice as high as the Peninsular subspecies. Our findings suggest the need for careful genetic management of both subspecies in order to minimize the further loss of genetic variability.