Bullseye snakehead, Channa marulius, was first detected in 2000 in the southern Florida town of Tamarac and has been expanding its geographic range. Environmental DNA (eDNA) analysis is a newly-developed technique used to non-invasively detect cryptic or low-density species, or those that are logistically difficult to study. Genetic material shed into the environment through tissue and body fluids is concentrated from water samples and analyzed for the presence of target species eDNA. To help delineate bullseye snakehead’s geographic range, we developed and validated a species-specific eDNA assay for both quantitative and droplet digital PCR (ddPCR). We then used ddPCR to assess 16 locations in southeast Florida using 222 water samples collected from 2015-2018. Positive eDNA detections were obtained at all six locations that were within the known geographic range of bullseye snakehead. Furthermore, eDNA was detected in six of 10 locations that were previously thought to be outside the periphery of the range but hydrologically connected through the extensive canal system. Over the four years of sampling, estimated occurrence rates (ψ) remained stable and relatively high (ψ = 0.67 [95% Confidence Interval (CI) 0.33-0.95]) near Tamarac, FL, as compared to the most southern sampling sites (ψ = 0.0-0.37). Bullseye snakehead eDNA estimated occurrence rates in the middle region increased between 2016 (0.28 [95% CI 0.03-0.94]) and 2017 (0.66 [95% CI 0.24-0.98]), potentially reflecting eDNA detections related to a growing or expanding population. Bullseye snakehead eDNA was detected at low concentrations on the northern and eastern borders of Everglades National Park, which is an important conservation area and UNESCO World Heritage Site. Despite extensive sampling via electrofishing, no bullseye snakeheads were visually detected in several locations that yielded positive eDNA samples. It is unclear whether eDNA was transported through flowing water or another vector. To date, collection records for this species are confined to urban canals; however, bullseye snakehead may use the interconnected system of canals to disperse to natural conservation areas, such as Everglades National Park, Big Cypress National Preserve and Water Conservation Areas, where it may impact native species via predation and competition.