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Aim: The conservation of biodiversity is hampered by data deficiencies, with many new species and subspecies awaiting description or reclassification. Population genomics and ecological niche modelling offer complementary new tools for uncovering functional units of phylogenetic diversity. We hypothesize that phylogenetically delineated lineages of gentoo penguins (Pygoscelis papua) distributed across Antarctica and sub‐Antarctic Islands are subject to spatially explicit ecological conditions that have limited gene flow, facilitating genetic differentiation, and thereby speciation processes. Location: Antarctica and sub‐Antarctic area. Methods: We identify divergent lineages for gentoo penguins using ddRAD‐seq and mtDNA, and generated species distribution models (SDMs) based on terrestrial and marine parameters. Results: Analyses of our genomic data supports the existence of four major lineages of gentoo penguin: (i) spanning the sub‐Antarctic archipelagos north of the Antarctic Polar Front (APF); (ii) Kerguelen Island; (iii) South America; and (iv) across maritime Antarctic and the Scotia Arc archipelagos. The APF, a major current system around Antarctica, acts as the most important barrier separating regional sister lineages. Our ecological analyses spanning both the terrestrial (breeding sites) and marine (feeding sites) realms recover limited niche overlap among the major lineages of gentoo penguin. We observe this pattern to correspond more closely with regional differentiation of marine conditions than to terrestrial macroenvironmental features. Main conclusions: Recognition of regional genetic lineages as discrete evolutionary entities that occupy distinct ecological niches and also differ morphologically should be considered a priority for conservation. Gentoo penguins provide a good example of how conservation policy can be directly impacted by new insights obtained through the integration of larger genomic datasets with novel approaches to ecological modelling. This is particularly pertinent to polar environments that are among the most rapidly changing environments on earth. [ABSTRACT FROM AUTHOR]

• Scarce genetic differentiation between Royal and macaroni penguins. • Population differentiation associated to distance between colonies and temperatures. • Macaroni penguins from Antarctic and sub-Antarctic exhibited genetic differentiation. The study of systematics in wide-ranging seabirds can be challenging due to the vast geographic scales involved, as well as the possible discordance between molecular, morphological and behavioral data. In the Southern Ocean, macaroni penguins (Eudyptes chrysolophus) are distributed over a circumpolar range including populations in Antarctic and sub-Antarctic areas. Macquarie Island, in its relative isolation, is home to a closely related endemic taxon — the royal penguin (Eudyptes schlegeli), which is distinguishable from E. chrysolophus mainly by facial coloration. Although these sister taxa are widely accepted as representing distinct species based on morphological grounds, the extent of their genome-wide differentiation remains uncertain. In this study, we use genome-wide Single Nucleotide Polymorphisms to test genetic differentiation between these geographically isolated taxa and evaluate the main drivers of population structure among breeding colonies of macaroni/royal penguins. Genetic similarity observed between macaroni and royal penguins suggests they constitute a single evolutionary unit. Nevertheless, royal penguins exhibited a tendency to cluster only with macaroni individuals from Kerguelen Island, suggesting that dispersal occurs mainly between these neighboring colonies. A stepping stone model of differentiation of macaroni/royal populations was further supported by a strong pattern of isolation by distance detected across its whole distribution range, possibly driven by large geographic distances between colonies as well as natal philopatry. However, we also detected intraspecific genomic differentiation between Antarctic and sub-Antarctic populations of macaroni penguins, highlighting the role of environmental factors together with geographic distance in the processes of genetic differentiation between Antarctic and sub-Antarctic waters. [ABSTRACT FROM AUTHOR]