Freshwater organisms in the Okavango Delta and Lake Ngami (Botswana) provide direct and indirect benefits to people and the economy of the region. However, their existence could be potentially threatened by human activities (primarily, upstream water abstraction and planned hydropower structures) coupled with climate change. For their protection, it is essential to know their distribution, ecology, and status of the ecosystems that they inhabit. Publications that record taxa from the Delta at species level are scarce, particularly aquatic macroinvertebrates. Identifying organisms to species level can provide more accurate information for environmental monitoring and conservation programmes but requires significant training and expertise. Here, we present a comprehensive taxonomical review of 2204 freshwater species from the Okavango Delta and Lake Ngami, with additional 355 species found in other areas of Botswana that are likely to be present in the study region. We also compare the diversity of the Okavango Delta and Lake Ngami with two other tropical wetlands: the Pantanal (Brazil) and the Kakadu Region (Australia). We show that biodiversity in the Okavango Delta and Lake Ngami is higher than in previous estimates, with recorded species richness dominated by phytoplankton and macroinvertebrates. Most species are widespread across the system and southern Africa. The resulting database includes new records (Bryozoa, Porifera), information on species conservation status, habitat, ecology, distribution in continental Africa, site details and taxonomical notes. This will be an essential resource for researchers, conservation managers, policy makers and consultants investigating freshwater biodiversity in tropical wetlands in the region.