In recent years, the degradation of biodiversity has advanced significantly, especially in freshwater ecosystems. To conserve rare species, the distribution of the target species should be known, even if the density is very low. Traditional habitat surveys using direct catches or observations require much time, labor, and expertise. Over the last decade, environmental DNA (eDNA) analysis methods that complement traditional surveys have been developed. In the present study, we developed an eDNA-based detection method for a Cyprinidae species, Hemigrammocypris rasborella, and applied it in natural habitats. First, we tested our method in 11 irrigation ponds for which information on the distribution of H. rasborella was available. The eDNA detection results matched completely with the known presence/absence data. Next, we applied this method to 81 irrigation ponds for which no distribution information was available, and detected the eDNA of H. rasborella in 6 ponds. Subsequently, we conducted capture surveys in the 6 eDNA-positive ponds and found the species in 5 ponds. These results suggest that eDNA analysis is useful for the monitoring of rare species.