The temporal components of genetic diversity and geographical structure of invasive mosquitofish populations are poorly known. Through the genetic monitoring of four consecutive cohorts of Gambusia holbrooki from three different river basins we aimed to determine temporal patterns of regional genetic variation and dispersal rates within invasive populations. Despite showing evidence of strong population size fluctuations, genetic diversity levels were maintained among local cohorts. We only detected temporal allele frequency changes associated with seasonal flooding that did not modify major trends on population structure among cohorts. Downstream gene flow coupled with increased connectivity at lowland locations to increase genetic diversity levels in these areas. A large proportion of local fish (up to 50 %) were dispersers, often originated from locations within the same river basin. High dispersal capability, ecological tolerance, and reproductive traits likely promote river colonization. Finally, our results also confirmed that human-assisted translocations promote within and among basin gene flow and maintained levels of genetic diversity, particularly in upstream locations

This work has been possible thank to the financial support of the former Spanish Ministry of Economy and Competitiveness (MINECO, CGL2009-12877-C02-02). DDM held a Ph.D. scholarship from the University of Girona (UdG, BR10/12)