Discerning the dispersal patterns of invasive species is critically important for the design of effective management strategies and the development of appropriate theoretical models predicting the spatial expansion of introduced populations. Post-introduction dispersal may occur naturally or via human transport, but for many organisms, assessing the relative contribution of each of these factors is difficult using traditional methods. Here, we explored the genetic patterns associated with the spread of red swamp crayfish (Procambarus clarkii) among 21 populations in the Pearl River basin and 2 peripheral populations in the Yangtze River basin. We found the genetic diversity of P. clarkii in the Pearl River basin was somewhat lower than in the Yangtze River basin. We also found (1) there was significant genetic differentiation between populations, (2) genetic differentiation was not related to geographic distance (i.e., isolation by distance), and (3) a Bayesian assignment analysis revealed three distinct genetic clusters and genetic admixture. Our results therefore provide evidence that human-mediated multiple introductions occurred in the Pearl River basin. Anthropogenic activities such as commercial transportation were likely responsible for the long-distance dispersal of P. clarkii. This study provides useful information for developing management strategies.