Freshwater eels of the genus Anguilla comprise 16 species that include three subspecies and are characterized by their unique catadromous life cycles. Their life histories and nocturnal life styles make it difficult to observe them in freshwater and marine habitats. To investigate their distribution and ecology in aquatic environments, we developed new PCR primers for metabarcoding environmental DNA (eDNA) from Anguilla. The new primers (MiEel) were designed for two conserved regions of the mitochondrial ATP6 gene, which amplify a variable region with sufficient interspecific variations ranging from five to 22 nucleotide differences (one to three nucleotide differences between three subspecies pairs). We confirmed the versatility of the MiEel primers for all freshwater eels using tissue DNA extracts when analyzed separately. The metabarcoding combined with the MiEel primers using mock communities enabled simultaneous detection of Anguilla at the species level. Analysis of eDNA samples from aquarium tanks, a controlled pond and natural rivers demonstrated that the MiEel metabarcoding could successfully detect the correct Anguilla species from water samples. These results suggested that eDNA metabarcoding with MiEel primers would be useful for non-invasively monitoring the presence of the endangered anguillid eels in aquatic environments where sampling surveys are difficult.