Since biodiversity is increasingly threatened in the Neotropics, the need for site-based, high-leverage conservation strategies oriented towards threat abatement and mitigation has become more and more urgent over the last decade. The development of a network of conservation action sites is a prerequisite for the formulation and implementation of such high-yield strategies. Therefore, an interdisciplinary team of scientific experts addressed the need for a network-based portfolio of biodiversity conservation action sites covering a total of 27 terrestrial and 5 marine ecoregions in Central America. During a first-iteration exercise action sites were identified, prioritized and mapped to establish a network-based portfolio applying the Ecoregional Planning (ERP) method. First, terrestrial, freshwater and coastal-marine conservation targets at multiple scales and levels of biological organization were selected, representing the whole range of biodiversity. This was done on basis of a gap analysis using existing topographic, geological and vegetation maps, digital elevation models (DEMs), and drainage system information. In combination with Ecological Land Units (ELUs), terrestrial targets (vegetation types) occurring along environmental gradients were used as surrogates for levels of biodiversity. Similarly, Ecological Drainage Units (EDUs) were applied to determine aquatic targets. Habitat types and ecological systems at the Pacific and/or Caribbean coast were selected as coastalmarine targets. A total of 403 terrestrial, 25 freshwater and 34 coastal-marine conservation targets were selected. GIS-based data analysis resulted in a series of thematic data layers and maps which were reviewed and validated by 46 experts on basis of target occurrences and viability. They identified a total of 143 areas of biodiversity significance including 78 terrestrial, 50 freshwater and 15 coastalmarine areas. To ensure adequate representation, a total of 10 occurrences per target were set as a minimum conservation goal. The final network-based portfolio captured 70% of the terrestrial, 56% of the freshwater and 84% of the coastal-marine goals and included 20 priority areas for immediate action: 3 in Belize, 4 in Guatemala, 3 in Honduras, 2 in Nicaragua, 3 in Costa Rica and 5 in Panama.