Although Chile has been establishing marine protected areas since the 1960s, studies to identify representative areas for marine biodiversity conservation at the ecoregional scale are lacking. Using the Marxan decision support tool we conducted a systematic planning process to identify High Conservation Value Areas in the channels and fjords of the southern Chile ecoregion. We involved 74 experts and other stakeholders in identifying conservation features, setting targets and compiling distribution data. Current distributions of 39 features were used in Marxan analyses. In one scenario we locked planning units that contained the Francisco Coloane Marine Protected Area. In a second scenario we also excluded planning units that overlapped with Appropriate Areas for Aquaculture, defined by the Chilean government's coastal zoning process. One-hundred percent of the proposed conservation targets were met in both scenarios. Although the distribution of 12 conservation features overlapped to a certain extent (>10%) with Appropriate Areas for Aquaculture, Marxan identified conservation solutions avoiding these areas. Our suggested portfolio of High Conservation Value Areas comprises a network of 33 sites, covering 99,432 km2 (12% of the ecoregion). These results provide the first science-based roadmap for decision makers and conservationists, and were used by the Chilean government in its coastal zoning process to define priority areas for conservation. Consequently, salmon farming has been excluded from Tierra del Fuego island.