Unimpeded transfer and spread of invasive species throughout freshwater systems is of global concern, altering species compositions, disrupting ecosystem processes, and diverting economic resources. The magnitude and complexity of the problem is amplified by the global connectedness of human movements and the multiple modes of inter-basin transport of aquatic invasive species. Our objective was to trace the fishing behavior of anglers delineating potential pathways of transfer of invasive species throughout the vast inland waters of the Great Lakes of North America, which contain more than 21% of the world’s surface freshwater and are among the most highly invaded aquatic ecosystems in the world. Combining a comprehensive survey and a spatial analysis of the movements of thousands of anglers in 12 states within the US portion of the Great Lakes Basin and the Upper Mississippi and Ohio River Basins, we estimated that 6.5 million licensed anglers in the study area embarked on an average of 30 fishing trips over the course of the year, and 70% of the individuals fished in more than one county. Geospatial linkages showed direct connections made by individuals traveling between 99% of the 894 counties where fishing occurred, and between 61 of the 66 sub-watersheds in a year. Estimated numbers of fishing trips to individual counties ranged from 1199–1.95 million; generally highest in counties bordering the Great Lakes. Of these, 79 had more than 10,000 estimated fishing trips originating from anglers living in other counties. Although angler movements are one mechanism of invasive species transfer, there likely is a high cumulative probability of invasive species transport by several million people fishing each year throughout this extensive freshwater network. A comprehensive georeferenced survey, coupled with a spatial analysis of fishing destinations, provides a potentially powerful tool to track, predict, curtail and control the transfer and proliferation of invasive species in freshwater.