This study presents a novel method for detecting species in freshwater using environmental DNA (eDNA). The approach involves using specific primers to amplify short mitochondrial DNA sequences, enabling the detection of species like the American bullfrog (Rana catesbeiana) in controlled environments and natural wetlands. The method was tested in controlled conditions and natural wetlands, with results showing reliable detection of the bullfrog even at low densities. The reliability of the results was confirmed through traditional sequencing and parallel pyrosequencing techniques. The study demonstrates that eDNA can be used to detect secretive organisms in wetlands without direct observation, offering a new perspective for biodiversity assessment from environmental samples. The method combines eDNA analysis with DNA barcoding and massive sequencing, enabling the identification of species from degraded DNA. The study highlights the potential of this approach for monitoring invasive species, threatened species, and other secretive aquatic organisms. The technique is particularly useful when traditional census methods are impractical or inaccurate. The study also discusses the challenges and limitations of eDNA analysis, including the potential for false positives and the need for careful sample handling. Overall, the study shows that eDNA can be a powerful tool for biodiversity assessment and species detection in aquatic environments.This study presents a novel method for detecting species in freshwater using environmental DNA (eDNA). The approach involves using specific primers to amplify short mitochondrial DNA sequences, enabling the detection of species like the American bullfrog (Rana catesbeiana) in controlled environments and natural wetlands. The method was tested in controlled conditions and natural wetlands, with results showing reliable detection of the bullfrog even at low densities. The reliability of the results was confirmed through traditional sequencing and parallel pyrosequencing techniques. The study demonstrates that eDNA can be used to detect secretive organisms in wetlands without direct observation, offering a new perspective for biodiversity assessment from environmental samples. The method combines eDNA analysis with DNA barcoding and massive sequencing, enabling the identification of species from degraded DNA. The study highlights the potential of this approach for monitoring invasive species, threatened species, and other secretive aquatic organisms. The technique is particularly useful when traditional census methods are impractical or inaccurate. The study also discusses the challenges and limitations of eDNA analysis, including the potential for false positives and the need for careful sample handling. Overall, the study shows that eDNA can be a powerful tool for biodiversity assessment and species detection in aquatic environments.