The alligator snapping turtle (Macrochelys temminckii) is under consideration for listing as a federally endangered species. Distributional data and estimates of population sizes are needed to make a sound decision regarding listing, but this information is largely unavailable due to the immense effort required for M. temminckii trapping surveys. To alleviate difficulty in detection and to help inform subsequent field-intensive survey efforts, we developed an environmental DNA (eDNA) method capable of providing presence data even in systems with high turbidity and suspended organic material. The assay we developed utilizes probe-based quantitative polymerase chain reaction and reliably amplifies M. temminckii eDNA in both lentic and lotic systems, with no amplification observed in other nontarget, sympatric turtle species. The novel eDNA method developed, optimized, and field-tested in this study provides a promising tool for detection of alligator snapping turtles, with resultant presence data likely to prove beneficial for the management and conservation of this species.