Environmental DNA (eDNA) detection is a rapidly expanding technique used to non-invasively detect cryptic, low density, or logistically difficult-to-study species, such as imperiled manatees. Genetic material shed into the environment through tissue and body fluids is concentrated from water samples and analyzed for the presence of targeted eDNA. To help delineate manatee habitat ranges, high use areas, and seasonal population changes, a cytochrome-b quantitative PCR and state-of-the-art droplet digital PCR (ddPCR) eDNA assay was developed for the three extant and vulnerable manatee species: both subspecies of the West Indian manatee (Florida and Antillean), the African manatee and Amazonian manatee. Occurrence (ψ) and detection (p) probabilities were estimated to inform management efforts and population monitoring. To validate the assay, water was analyzed from a relatively high-density Florida manatee east coast population and produced an average 31,564 target DNA molecules/liter (ψ=0.84 (0.40-0.99); p=0.99 (0.95-1.00)). Similar occupancy estimates were produced from investigations of less well-characterized Florida manatee populations in the Florida Panhandle (ψ=0.79 (0.54-0.97)) and Cuba (ψ=0.89 (0.54-1.00)) while occupancy estimates of the African species in Cameroon were lower (ψ=0.49 (0.09-0.95)). The estimates were higher than those generated using aerial survey data on the west coast of Florida. Future eDNA studies could assess locations where manatees are difficult to identify visually (e.g., dark or turbid water common in the Amazon River, and Africa), are present in patchy distributions, or where repatriation efforts are proposed (e.g., Brazil, Guadeloupe). Moreover, this technology could be extended to species on the verge of extinction (e.g., manatees in Jamaica and Haiti, and Asian dugongs), where conventional survey methods are challenging.