A protected areas system (PAS) is effective only when it adequately includes a representative sample of important geophysical and biological features, including critically endangered biota of a region. However, protected areas in Nepal, as in many parts of the world, have been established on an ad hoc basis, and thus one or more important features have been overlooked. We conducted a gap analysis and developed a comparison index to assess the representativeness of geophysical features (physiography, altitude, and ecoregions), species diversity, and endangered species listed in International Union for Nature Conservation (IUCN) and Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) by using a Geographical Information System (GIS) framework. The analysis indicated that more than two thirds (67.84%) of the total area of protected areas (PAs) is in high mountains, although this region accounts for only 23.92% of the country's total area. The hills comprise the highest proportion (29.17%) of the country's area but currently have the smallest proportion (1.33%) of PAs. The altitudinal zones between 200–400 m are well represented with PAs; however, the region between 400–2700 m is poorly represented, and representation by PAs is comparatively higher in the area above 2800 m. The ecoregions that have high conservation priority at global scale are poorly represented in protected areas of Nepal. Existing PAs include 39.62% of flowering plants, 84.53% of mammals, 95.73% of birds, and 70.59% of herpetofauna of the country. Threatened animal species are well protected, whereas a large number of threatened plant species are not represented by the current PA system.