Clarifying the intricate history of unrecorded fish invasions represents an important step in understanding the invasion process. Here, we elucidate the invasion of a Neotropical river basin in Southeastern Brazil by a very efficient predator, the white piranha (Serrasalmus brandtii). We used a temporal series of population dynamics data (2008–2016) and analyses of mitochondrial DNA sequences (COI, 16S, and control region) of specimens from the entire native (Sao Francisco River Basin, Southeastern Brazil) and invaded (Jequitinhonha River Basin, JRB) ranges. We detected low genetic diversity (h = 0.5835) and strong genetic structure in the invasive range (Fst = 0.9141), despite high genetic diversity (h = 0.8900) and low genetic structure (Fst from 0.0740 to 0.1348) in the native range. High genetic structure in the invaded range was associated with a hydroelectric dam that prevented the admixture of two independent introductory acts into the JRB (downstream and upstream of the Irape Dam). The rapid invasion capability of Serrasalmus brandtii, with few propagules, indicates that the species should be included in ecological risk assessments for restocking efforts of other commercially or ecologically important fish species and dam construction in Brazil. The combination of genetic and population dynamic datasets enabled the reconstruction of a top predator fish invasion in the Neotropics and shed light on ecological factors that influenced its invasion success.