This document provides metadata on the BioMatrix (BioFresh Task 4.4) to guide usage. The BioMatrix is an output for the BioFresh project funded by the European Union under the Seventh Framework Programme (FP7), Theme 6 (Biodiversity of Freshwater Ecosystems: Status, Trends, Pressures and Conservation Priorities). The BioMatrix is a data repository of contemporary distributions of freshwater biodiversity created by the BioFresh project, largely drawn from species range maps, a range of environmental variables, species trait data, and IUCN Red List TM species assessments. A particular advantage of the BioMatrix is that it enables spatial analyses within and between regions across the globe as, for the first time, it provides all the data in a common spatial framework, i.e. all data are mapped to the HydroBASINS1 catchments, also created as part of the BioFresh project. In addition, the data will be available at other spatial resolutions such as WWF’s freshwater ecoregions.