Biophysical characterization analyses of protected areas (PA) that provide information on their ecological values and potential areas with similar characteristics are needed to make informed PA network planning and management decisions. This study combines and further develops methodologies that use remote sensing and modelling to identify habitat functional types in PAs and map similar areas at the ecoregion level. The study also develops new terrestrial habitat diversity and irreplaceability indices at habitat and PA scale that allow the comparison and ranking of PAs in terms of biophysical gradients and singular environmental conditions. Six PAs were selected to highlight and discuss the results of the proposed methodology. Both individual and composite indices should be considered when trying to compare PAs to understand the overall complexity and ecological values of each PA. Results can inform planning and management of individual and protected area networks as well as identify new areas for conservation. The information provided by the model about similar habitats outside protected areas can also help assess their representativeness and support studies to strengthen ecological connectivity. Besides systematic comparisons, detailed assessments of protected areas can also be performed using medium and high-resolution input variables. This is especially relevant for protected areas in developing countries where undertaking fieldwork is very difficult and the budget devoted to conservation is limited.