Genetic analyses contribute to studies of biological invasions by mapping the origin and dispersal patterns of invasive species occupying new territories. Using microsatellite loci, we assessed the genetic diversity and spatial population structure of mosquitofish (Gambusia holbrooki) that had invaded Spanish watersheds, along with the American locations close to the suspected potential source populations. Mosquitofish populations from the Spanish streams that were studied had similar levels of genetic diversity to the American samples; therefore, these populations did not appear to have undergone substantial losses of genetic diversity during the invasion process. Population structure analyses indicated that the Spanish populations fell into four main clusters, which were primarily associated with hydrography. Dispersal patterns indicated that local populations were highly connected upstream and downstream through active dispersal, with an average of 21.5% fish from other locations in each population. After initially introducing fish to one location in a given basin, such dispersal potential might contribute to the spread and colonization of suitable habitats throughout the entire river basin. The two-dimension isolation-by-distance pattern here obtained, indicated that the human-mediated translocation of mosquitofish among the three study basins is a regular occurrence. Overall, both phenomena, high natural dispersal and human translocation, favor gene flow among river basins and the retention of high genetic diversity, which might help retain the invasive potential of mosquitofish populations