Abstract The Delmarva fox squirrel (Sciurus niger cinereus) has been listed as endangered by the United States Department of Interior since 1967. A high-priority task for the recovery of this taxon is to determine its current geographic distribution. Toward this end, we have identified a microsatellite locus that unambiguously differentiates Delmarva fox squirrels from eastern gray squirrels (S. carolinensis), which frequently co-occur with Delmarva fox squirrels. Analysis of this marker in noninvasively collected hair samples will allow unequivocal identification of localities occupied by Delmarva fox squirrels with a minimum investment of funds, time, and effort because handling individuals will be unnecessary. This protocol will expedite site review in connection with the Endangered Species Act consultation process.