Resolving the genetic connectivity of coral reef taxa is necessary to understand the community dynamics of these increasingly threatened ecosystems. Herein, we assess the fine scale genetic connectivity of six populations of the Atlantic giant barrel sponge, Xestospongia muta (Schmidt, 1870), using microsatellite markers. This survey included populations from across the Florida Reef Tract and the Gulf of Mexico, including sponges from the Pulley Ridge Habitat Area of Particular Concern, a mesophotic coral reef located approximately 250 km from Florida’s (USA) southwestern coast, and the Flower Garden Banks National Marine Sanctuary, the northernmost western Atlantic coral reef ecosystem. Overall, significant population structure was found (F ST = 0.020, 95% CI: 0.011–0.031), with X. muta individuals from the Flower Garden Banks showing the highest levels of genetic differentiation relative to all other surveyed populations (F ST > 0.048). Despite the high levels of population structuring observed, some horizontal and/ or vertical connectivity was found between neighboring reef systems, including evidence of gene flow between mesophotic (Pulley Ridge Habitat of Particular Concern) and photic (Dry Tortugas) reef tracts. Furthermore, largely negligible levels of first-generation migration among discrete genetic populations was observed, suggesting that the persistence of most populations of X. muta is highly dependent on self-recruitment. Thus, while the Pulley Ridge mesophotic reef may provide sponge recruits to shallow photic reefs, its role as a potential refuge and propagule source appears limited to only small geographic scales.