•Conservation of freshwater fishes is dependent on accurate and precise data.•Environmental DNA analysis has the potential to improve species conservation.•eDNA can improve sampling effectiveness and efficiency compared to capture sampling.•This review describes eDNA analysis as it relates to fisheries conservation. Conservation of fishes is dependent on accurate and precise data about the distributions and population status of at-risk species. Moreover, effective management of fisheries requires that data be collected in ecologically and politically actionable timeframes. Environmental DNA (eDNA) analysis, the extraction and identification of DNA from environmental samples, is a relatively new bioassessment method with the potential to improve species detection probabilities and efficiency compared to traditional capture or observation-based sampling approaches. Here, we provide a primer on eDNA analysis as it pertains to fisheries ecology and conservation. We provide a summary of the history and current status of eDNA analysis with particular attention paid to the study of fishes. We highlight the major advances that have transformed eDNA analysis into an application-ready tool that can assist fisheries professionals in achieving research and conservation goals. Furthermore, we provide an overview of the limitations of eDNA as they pertain to fisheries science.