The effective management of rare and threatened species, especially in areas where population sizes have diminished, relies on knowledge of their distribution. This is a particular challenge in aquatic environments for cryptic species with low abundance. Environmental DNA (eDNA) approaches offer improved detection rates of rare and threatened species in comparison with traditional sampling approaches. In this study, eDNA sampling and traditional seine netting were undertaken across the historical range of the Critically Endangered estuarine pipefish (Syngnathus watermeyeri) in South Africa. Across 39 sites, 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 potential relationships with pipefish presence. We successfully detected S. watermeyeri in two estuaries (Kariega and Bushmans) using both methods. The lack of detections in the Kasouga, East Kleinemonde and West Kleinemonde estuaries, adds to the growing body of evidence that the estuarine pipefish has experienced local extinctions in its former range and is now found in just two estuarine systems. Importantly, the rate of detection using eDNA was more than four times (0.43) that of seine netting (0.1). Results confirm that the occurrence of S. watermeyeri is highly dependent on the presence of dense beds of submerged macrophytes like Zostera capensis (eelgrass). This work further validates the use of species-specific qPCR assays and provides the foundation for developing a highly sensitive long-term monitoring program for this Critically Endangered species.