The authors present a novel approach to detect the presence of a species in freshwater environments using environmental DNA (eDNA). They used specific primers to amplify short mitochondrial DNA sequences of the American bullfrog (Rana catesbeiana) in controlled and natural wetland environments. The method successfully detected the species in all environments where it was present, even at low densities, through traditional sequencing and parallel pyrosequencing techniques. The reliability of the results was demonstrated by the identification of amplified DNA fragments. This approach, combined with massive sequencing and DNA barcoding, opens new perspectives for assessing current biodiversity from environmental samples, particularly for secretive or invasive species. The study highlights the potential of eDNA for detecting species presence without direct observation, which is valuable for biodiversity studies, conservation biology, and ecology.The authors present a novel approach to detect the presence of a species in freshwater environments using environmental DNA (eDNA). They used specific primers to amplify short mitochondrial DNA sequences of the American bullfrog (Rana catesbeiana) in controlled and natural wetland environments. The method successfully detected the species in all environments where it was present, even at low densities, through traditional sequencing and parallel pyrosequencing techniques. The reliability of the results was demonstrated by the identification of amplified DNA fragments. This approach, combined with massive sequencing and DNA barcoding, opens new perspectives for assessing current biodiversity from environmental samples, particularly for secretive or invasive species. The study highlights the potential of eDNA for detecting species presence without direct observation, which is valuable for biodiversity studies, conservation biology, and ecology.