|Abstract:||The introduction, establishment, and spread of invasive species has had many irreparable negative effects on ecosystems and causes significant economic harm. Crayfishes are a diverse group of freshwater crustaceans which have proven to be harmful invasive species throughout the world. Additionally, invasive crayfishes are a leading cause of displacement of native crayfishes. Detecting invasive species and determining their distribution is critical to assessing risks to ecosystems and selecting effective management and control options. In this study, we wanted to determine the distribution of non-native Faxonius virilis in the Ozark National Scenic Riverways, Missouri, U.S.A., where it has been spreading since the 1980s. Additionally, we wanted to develop an environmental DNA (eDNA) assay to aid in detection of F. virilis. While developing the F. virilis-specific eDNA assay, we noticed a discordance between the phenotype and mitochondrial DNA barcode of some native F. punctimanus specimens. As evidenced by a mismatch in mitochondrial, phenotypic, and microsatellite data, we have found that non-native F. virilis have hybridized with native F. punctimanus in the Ozark National Scenic Riverways. Hybridization has rarely been documented between crayfish species and before this study, the only genetically documented reports of North American crayfish species hybridizing have been between invasive F. rusticus and two congeners. Traditional sampling outperformed eDNA in our system and hybridization was not supported by modelling as a factor influencing eDNA detections. While it did not perform well for detecting F. virilis in the Current River watershed, eDNA remains an important tool. Overall, our results support previous researchers’ remarks that undocumented hybridization between native and non-native crayfish may be more common than previously thought.