AbstractThe effective management of rare and threatened species, especially in areas where population sizes have diminished, relies on knowledge of their population size, threats, and distribution. Robust mapping of distribution presents a particular challenge in aquatic environments for cryptic species, especially those with low abundance. Environmental DNA (eDNA) approaches can offer improved detection rates of many rare and threatened species when compared with traditional sampling approaches. In this study, we developed and optimized a targeted eDNA assay for the critically endangered estuarine pipefish (Syngnathus watermeyeri). eDNA sampling and seine netting were undertaken at 39 sites across the historical range of S. watermeyeri in the Eastern Cape of South Africa in 2019. At each site, five water samples were collected for eDNA analysis (n = 195) along with three seine netting hauls (n = 117). Habitat and environmental data were collected at each location to explore what physical and biotic parameters might correlate with pipefish presence/absence. We successfully detected S. watermeyeri in two estuaries (Kariega and Bushmans) using both survey methods. Importantly, the positive detection rate of eDNA (66.7%) was four times that of seine netting (16.7%), highlighting the value of eDNA as a monitoring tool for rare and cryptic species. Null detections in the Kasouga, East Kleinemonde, and West Kleinemonde estuaries add to the growing body of evidence that the estuarine pipefish has been extirpated from these locations and is now only found in two estuarine systems. The occurrence of S. watermeyeri was found to be highly dependent on the cover of submerged macrophytes such as Zostera capensis (eelgrass). By providing a more complete picture of the conservation status of this critically endangered species, this work facilitates the development of a long‐term monitoring program and the identification of priority conservation areas.