Aquatic invasive species (AIS) are recognized globally as a major threat to native ecosystems and are associated with a high economic impact and difficulty in eradication once established. Prevention is the most effective approach to minimizing negative effects of AIS, but new introductions of AIS continue to occur each year in the United States indicating that current prevention measures are ineffective or non-existent. To develop comprehensive management recommendations, we conducted a gap analysis of invasive species’ primary introduction pathways to determine and quantify where prevention measures are lacking. Using the U.S. Geological Survey’s Nonindigenous Aquatic Species (NAS) database we identified 17 AIS representative of 8 taxonomic groups and 8 introduction pathways to serve as case studies outlining the current state of prevention. Five federal agencies (USFWS, USDA, EPA, USCG, and NOAA) were identified as having authority over AIS primary prevention. Management gaps were identified to varying degrees, most commonly via weak/ambiguous authority or regulation, for all pathways examined including hull/biofouling, ballast water, aquaculture, the live food market (including prayer release), the aquarium/pet trade, ornamental plant trade, and detection of hitchhikers. Federal managers were surveyed to assess the “ideal” vs “practical” state of prevention. We found that manger’s opinions shifted an average of 23% away from agreement when characterizing what management strategies would be “ideal” vs “practical”. NAS database records also demonstrated that the aquarium/pet trade pathway is the largest contributor of species in the database. Additionally, the plant and freshwater fish taxonomic groups contributed the most species and records compared to any other database groups. Our combined quantification of pathway contributions to AIS introduction with increased understanding of existing gaps in prevention will directly aid 13 federal agencies as well as state and local governments with important implications for AIS prevention on a national scale.