The relative importance of multiple vectors to the initial establishment, spread, and population dynamics of invasive species remains poorly understood. This study used molecular methods to clarify the roles of commercial shipping and recreational boating in the invasion by the cosmopolitan tunicate, Botryllus schlosseri. We evaluated i) single vs. multiple introduction scenarios, ii) the relative importance of shipping and boating to primary introductions, iii) the interaction between these vectors for spread (i.e., the presence of a shipping-boating network), and iv) the role of boating in determining population similarity. Tunicates were sampled from 26 populations along the Nova Scotia, Canada, coast that were exposed to either shipping (i.e., ports), or boating (i.e., marinas) activities. A total of 874 individuals (~30 per population) from 5 ports and 21 marinas was collected and analyzed using both mitochondrial cytochrome c oxidase subunit I gene (COI) and 10 nuclear microsatellite markers. The geographical location of multiple hotspot populations indicates that multiple invasions have occurred in Nova Scotia. A loss of genetic diversity from port to marina populations suggests a stronger influence of ships than recreational boats on primary coastal introductions. Population similarity analysis reveals a clear dependence of marina populations on those that had been previously established in ports and connectivity due to a boating network better explains patterns in population similarities than does natural spread. We conclude that frequent primary introductions arise by ships and that secondary spread occurs gradually thereafter around individual ports, facilitated by recreational boating.