ABSTRACT: Environmental DNA (eDNA) has the potential to play an important role in surveys for rare and endangered aquatic species. Environmental DNA sampling is non-invasive and, for small cryptic species that are difficult to survey, it may offer a viable, more efficient, and less expensive alternative than traditional techniques. We used eDNA for surveying 5 endangered spring-endemic species at Bitter Lake National Wildlife Refuge, Chaves County, New Mexico, USA. Water samples (n = 40) in spring systems were evaluated in July 2018 for presence of residual DNA for Gambusia nobilis, Gammarus desperatus, Juturnia kosteri, Pyrgulopsis roswellensis, and Assiminea pecos. We detected eDNA at 50% of sites for G. nobilis, 42.5% of sites for J. kosteri, 27.5% of sites for P. roswellensis, 20% of sites for G. desperatus, and did not detect A. pecos eDNA at any of the sites. We also examined the habitat conditions that drive patterns in occupancy for these endangered species and developed thresholds for habitat parameters to inform conservation decisions. Salinity and dissolved oxygen influenced sample occupancy for G. desperatus, P. roswellensis, and J. kosteri, but only dissolved oxygen influenced sample occupancy for G. nobilis. Results highlighted the efficacy of using eDNA to monitor 4 of the 5, spring-endemic species, and provided insight to the habitat preferences for each species, which will help drive conservation activities.