Streams in the U.S. Virgin Islands (USVI; St. Croix, St. John, and St. Thomas), known locally as "guts", historically exhibited perennial flows, which have diminished substantially in recent decades due to a number of factors. While an abundance of anecdotal evidence suggests guts once supported robust communities across the USVI, to date no study has sought to quantify them. This knowledge gap is a barrier to conservation and management efforts in the USVI as little information is known about the abundance and distribution of potential species of greatest conservation need (SGCNs). Moreover, human-made barriers to aquatic organism passage may also limit recolonization of critical habitats by SGCNs from the Caribbean Sea after drought/flood events. To fill this gap, we surveyed over 50 locations across the three islands for streamflow, physical and chemical habitat, and biological communities using backpack electrofishing. All native freshwater fish and decapod species known to the USVI have been detected in data collection to date. Analyses are in progress, and species distribution and occurrence are expected to illustrate habitat fragmentation caused by anthropogenic barriers among other habitat variables such as water quality, elevation, and land use. This study will provide a freshwater connectivity plan and the first comprehensive survey of freshwater fauna distribution in the USVI.