The Humboldt penguin, once common throughout its range, is today listed as Vulnerable by the IUCN. Mark-recapture and telemetry studies indicate that adult Humboldt penguins are sedentary, suggesting strong genetic differentiation between colonies. We developed genotypes for 336 individuals at 12 microsatellite loci sampled at four different localities spanning the entire range of this species. Results show that long-term gene flow has occurred but appears to be affected by geographic distance as pairwise F ST comparisons involving the colony at Punta San Juan (Peru) and the two colonies at Algarrobo (central Chile) and Puñihuil (southern Chile) are significant. Bayesian estimates of recent migration rates indicate substantial dispersal among all colonies. Despite the dramatic decline in numbers, we did not observe a bottleneck in any population. Furthermore, we did not detect a founder effect in the recently discovered colony at Puñihuil. As our indirect estimates signal strong gene flow between populations, we suggest that Humboldt penguin colonies need to be managed as a metapopulation rather than as discrete management unit