Black Carp (Mylopharyngodon piceus) was imported to the USA to control aquaculture pond snails. This species has escaped captivity and occurs in parts of the Mississippi River, several tributaries, and floodplain lakes, which is concerning due to potential competition with native fishes and predation on native mussels, many of which are imperiled. However, Black Carp captures have primarily been incidental by commercial fishers, and evidence of reproduction in the wild is limited. The objectives of this study were to assess relative abundance of aquaculture-origin and wild Black Carp using ploidy and otolith stable isotope analysis, identify spatial extent of natural reproduction using otolith microchemistry, assess age distributions of wild and aquaculture-source Black Carp to infer years in which natural reproduction occurred and timing of aquaculture escapement or introductions, and estimate size and age at maturation to assess whether recruitment to adulthood has occurred. Results revealed that Black Carp are established in parts of the Mississippi River basin based on findings that: (1) non-captive Black Carp primarily consist of fertile, naturally-reproduced fish, (2) reproduction has occurred in several rivers, (3) multiple year classes of wild fish are present, and (4) wild fish have recruited to adulthood. Multiple introductions or escapements of aquaculture-source fish into the wild, including both fertile and functionally sterile individuals, were also inferred. Individual growth appears to be rapid, although considerable variation was observed among fish. Additional study is suggested to refine understanding of where and when Black Carp reproduction is occurring in the Mississippi River basin.