Coupled bio-physical models of larval dispersal predict that the Costa Rica–Panama (CR–PAN) reefs should constitute a demographically isolated region in the western Caribbean. We tested the hypothesis that CR–PAN coral reef fish populations would be isolated from Mesoamerican Barrier Reef System (MBRS) populations. To test that, we assessed population genetic structure in bicolor damselfish (Stegastes partitus) from both regions. Adult fish were genotyped from five reefs in CR–PAN and from four reefs along the MBRS at 12 microsatellite loci. Between-region F ST (F ST = 0.0030, P < 0.005) and exact test (x 2 = 74.34, df = 18, P < 0.0001) results indicated that there is weak but significant genetic differentiation between regions, suggesting some restriction in connectivity along the Central American coastline, as predicted by bio-oceanographic models. Additionally, there is among-site genetic structure in the CR–PAN region, relative to the MBRS and between regions, suggesting higher self-recruitment within CR–PAN. This finding may be explained by differences in habitat characteristics.