Habitat destruction and anthropogenic drivers have led to amphibian population declines worldwide, but the conservation status of many species remains in question. This study reports on the distribution of Mudpuppies, Necturus maculosus, in southeast Ohio, where widespread acid mine drainage and other forms of habitat destruction have led to severe declines and extinction in many waterways. Within the last century, however, the region has reforested, and damage to some streams has been mitigated, providing opportunities for Mudpuppy recolonization and population recovery. However, being a relatively secretive species, Mudpuppies require difficult and time intensive field surveys to detect. Therefore, the current distribution and conservation status of Mudpuppies in southeast Ohio is unclear. As a first step in documenting the current distribution and abundance of Mudpuppies in southeast Ohio, we conducted a rapid species assessment using environmental DNA (eDNA) surveys (September–November 2016) at 10 stream sites. We detected Mudpuppies at six of 10 streams using eDNA, including four streams in which they were known to occur from historical records and two streams from which Mudpuppies had not been previously reported. We also collected habitat data at each site, including concentrations of heavy metals and nutrients, physical stream habitat, conductivity, pH, temperature, total dissolved solids, and oxygen levels. Using logistic regression, we found composite Qualitative Habitat Evaluation Index (QHEI) scores were the best predictor of Mudpuppy presence. Our results suggest Mudpuppy eDNA is not easily detected when they are at low density, and animals may need to be within approximately 182 m of sampling points to be detected.