Environmental DNA (eDNA) detection of invasive species can be used to delimited occupied ranges and estimate probabilities to inform management decisions. Environmental DNA is shed into the environment through skin cells and bodily fluids and can be detected in water samples collected from lakes, rivers, and swamps. In south Florida, invasive Burmese pythons occupy much of the Greater Everglades in mostly inaccessible habitat and are credited with causing severe declines of native species’ populations. Detection of Burmese pythons by traditional methods, such as trapping and visual searching, have been largely ineffective, making eDNA a superior method for differentiating invaded habitat. We adapted a quantitative PCR eDNA assay for droplet digital PCR, a state-of-the-art method that improves precision and accuracy. From August 2014 to October 2016, locations in and around Arthur R. Marshall Loxahatchee National Wildlife Refuge in southeast Florida were surveyed for Burmese python eDNA. The Refuge is maintained to provide water storage and is considered one of the last remnants of the northern Everglades wetlands. Positive eDNA detections were made at each of the five sampling events, assessing a total of 399 samples, with moderate occurrence (ψ=58-91%) and detection (p=40-70%) probabilities, potentially reduced by high PCR inhibition-levels. The high occurrence rates and geographic distribution of the positive samples within the Refuge suggests a steady release of python eDNA from a resident Burmese python population and reduces support for primarily transport of eDNA through boats or flowing water from the north. The first confirmed sighting of a Burmese python in the Refuge occurred in September 2016, after eDNA testing had indicated the presence of pythons. An established population is not expected this far north, however, the detections likely indicate northern range limit of a consistent population at Loxahatchee on the eastern side of the Florida peninsula. Our study demonstrates the benefit of eDNA for determining more accurate range limits and expansion information for Burmese pythons, as well as laying the foundation for the assessment of control efforts.