The upwelling hypothesis has been proposed to explain reduced or lack of population structure in seabird species specialized in food resources available at cold-water upwellings. However, population genetic structure may be challenging to detect in species with large population sizes, since variation in allele frequencies are more robust under genetic drift. High gene flow among populations, that can be constant or pulses of migration in a short period, may also decrease power of algorithms to detect genetic structure. Penguin species usually have large population sizes, high migratory ability but philopatric behavior, and recent investigations debate the existence of subtle population structure for some species not detected before. Previous study on Humboldt penguins found lack of population genetic structure for colonies of Punta San Juan and from South Chile. Here, we used mtDNA and nuclear markers (10 microsatellites and RAG1 intron) to evaluate population structure for 11 main breeding colonies of Humboldt penguins, covering the whole spatial distribution of this species. Although mtDNA failed to detect population structure, microsatellite loci and nuclear intron detected population structure along its latitudinal distribution. Microsatellite showed significant Rst values between most of pairwise locations (44 of 56 locations, Rst = 0.003 to 0.081) and 86% of individuals were assigned to their sampled colony, suggesting philopatry. STRUCTURE detected three main genetic clusters according to geographical locations: i) Peru; ii) North of Chile; and iii) Central-South of Chile. The Humboldt penguin shows signal population expansion after the Last Glacial Maximum (LGM), suggesting that the genetic structure of the species is a result of population dynamics and foraging colder water upwelling that favor gene flow and phylopatric rate. Our findings thus highlight that variable markers and wide sampling along the species distribution are crucial to better understand genetic population structure in animals with high dispersal ability.