Globally distributed, the bottlenose dolphin (Tursiops truncatus) is found in a range of offshore and coastal habitats. Using 15 microsatellite loci and mtDNA control region sequences, we investigated patterns of genetic differentiation among putative populations along the eastern US shoreline (the Indian River Lagoon, Florida, and Charleston Harbor, South Carolina) (microsatellite analyses: n = 125, mtDNA analyses: n= 132). We further utilized the mtDNA to compare these populations with those from the Northwest Atlantic, Gulf of Mexico, and Caribbean. Results showed strong differentiation among inshore, alongshore, and offshore habitats (ФST = 0.744). In addition, Bayesian clustering analyses revealed the presence of 2 genetic clusters (populations) within the 250 km Indian River Lagoon. Habitat heterogeneity is likely an important force diversifying bottlenose dolphin populations through its influence on social behavior and foraging strategy. We propose that the spatial pattern of genetic variation within the lagoon reflects both its steep longitudinal transition of climate and also its historical discontinuity and recent connection as part of Intracoastal Waterway development. These findings have important management implications as they emphasize the role of habitat and the consequence of its modification in shaping bottlenose dolphin population structure and highlight the possibility of multiple management units existing in discrete inshore habitats along the entire eastern US shoreline.