The Humboldt penguin (Spheniscus humboldti) was once very common throughout its range along the coast of Peru and Chile. Today, listed as endangered, it is crucial to gain an understanding of gene flow and levels of genetic variation between breeding colonies to protect this species effectively. We developed seven microsatellite primers to investigate gene flow and population structure among four colonies. Locus-specific allelic diversity ranges from five to 11 alleles and observed heterozygosity ranges from 0.50 to 0.88. These markers cross-amplify in eight penguin species over five genera.