The Black Carp, Mylopharyngodon piceus, is an increasingly widespread invasive species in North America that threatens freshwater mussel populations. We developed four qPCR assays for detecting environmental DNA (eDNA) from these Black Carp populations. Assays were designed to target four mitochondrial DNA loci and were based on 34 complete mitochondrial genome sequences, including 29 generated in this study from samples obtained in three countries. Assays were validated for taxon specificity with in silico comparisons against archived DNA sequences and with in vitro tests of 41 DNA samples from Black Carp, as well as DNA samples from 30 non‐target fish species, all from the Mississippi River Basin. All four assays were able to detect the DNA of all Black Carp samples and did not exhibit any positive results with DNA from other tested species. Tests conducted in round‐robin fashion among three different laboratories found that all four assays were able to detect DNA at very low template concentrations (limits of detection = 3 copies/qPCR, limits of quantification = 16‐64 copies/qPCR) and, as part of in situ validation, were successful in detecting eDNA from Black Carp in aquaculture ponds. Despite some challenges with other attempts at in situ validation, the assays were also effective in detecting Black Carp eDNA in water samples from a drainage ditch in the upper reaches of the species’ range that was known to contain juvenile Black Carp, as well as in water samples from the Missisippi River and a connected oxbow lake in the lower reaches of the species range.