We assessed colony- and island-level genetic differentiation for the flightless cormorant (Phalacrocorax harrisi), an endangered Galápagos endemic that has one of the most limited geographical distributions of any seabird, consisting of only two adjacent islands. We screened 223 individuals from both islands and nine colonies at five microsatellite loci, recovering 23 alleles. We found highly significant genetic differentiation throughout the flightless cormorant's range on Fernandina and Isabela Islands (global FST = 0.097; P < 0.0003) both between islands (supported by Bayesian analyses, FST and RST values) and within islands (supported only by FST and RST values). An overall pattern of isolation-by-distance was evident throughout the sampled range (r = 0.4169, one-sided P ≤ 0.02) and partial Mantel tests of this relationship confirmed that ocean is a dispersal barrier (r = 0.500, one-sided P ≤ 0.003), especially across the 5-km gap between the two islands. The degree of detected genetic differentiation among colonies is surprising, given the flightless cormorant's limited range, and suggests a role for low vagility, behavioural philopatry, or both to limit dispersal where physical barriers are absent. We argue that this population should be managed as at least two genetic populations to better preserve the species-level genetic diversity, but, for demographic reasons, advocate the continued conservation of all breeding colonies.