The use of DNA from fecal samples can allow for a better understanding of the ecology of a species without capturing and handling the animals. This is particularly useful for cryptic and elusive animals, such as bats. Being able to identify critical habitat, such as maternity roosts, for bat species in areas where they depend on abandoned mines that could be slated for closure is necessary to enact appropriate protections for such roosts. In particular, Corynorhinus townsendii commonly uses abandoned mines for maternity roosts. Further, maternity roosts are difficult to identify through visual surveys when only one or a few surveys are performed before mine closures. We have developed a method for identification of C. townsendii maternity roosts that uses fecal DNA extracted from fresh guano collected from plastic sheeting placed at a mine entrance. We provide a multiplex PCR assay to amplify a control region fragment found only in C. townsendii, as well as a Y-linked protein (DBY) to detect male C. townsendii DNA. The purpose of this study was to identify a temporal shift in the presence of male C. townsendii bats, which can be useful to identify a maternity roost. This method allows for noninvasive identification of critical habitat for this species and reduces the effort and safety risk of entering mines on the part of biologists.