AbstractAim To delineate biogeographical patterns in Galapagos shallow‐water reef fauna at regional scales.Location Galapagos Islands.Methods Fishes and macro‐invertebrates were quantitatively censused using underwater visual techniques along more than 500 transects at defined depth strata across the Galapagos archipelago. Data were analysed using multivariate techniques to define regional patterns and identify species typical of different regions.Results Subtidal communities of fishes and macro‐invertebrates on shallow reefs differed consistently in species composition across the Galapagos archipelago, with three major biogeographical groupings: (1) the ‘far‐northern area’ containing the islands of Darwin and Wolf, (2) the ‘central/south‐eastern area’, including the east coast of Isabela, and (3) the ‘western area’, encompassing Fernandina and western Isabela. In addition, the northern islands of Pinta, Marchena and Genovesa form a separate region in the central/south‐eastern area, and Bahia Elizabeth and Canal Bolivar separate from other parts of the western area. The far‐northern bioregion is characterized by high fish species richness overall, including a high proportion of species of Indo‐Pacific origin. However, very few endemic fishes or species with distributions extending south from Ecuador (‘Peruvian’ species) are present, and the bioregion also possesses relatively low species richness of mobile macro‐invertebrate taxa. By contrast, the ‘western’ bioregion possesses disproportionately high numbers of endemic fish taxa, high numbers of cool‐temperate Peruvian fish species, and high invertebrate species richness, but very few species of Indo‐Pacific origin. The Bahia Elizabeth/Canal Bolivar bioregion possesses more endemic species and fewer species with Peruvian affinities than coasts within the western bioregion. The northern bioregion of Pinta, Marchena and Genovesa represents an overlap zone with affinities to both the far‐northern and south‐eastern islands. The south‐eastern bioregion includes species from a variety of different sources, particularly ‘Panamic’ species with distributions extending north to Central America.Main conclusions On the basis of congruent divisions for reef fish and macro‐invertebrate communities, the Galapagos archipelago can be separated into three major biogeographical areas, two of which can be further subdivided into two regions. Each of these five bioregions possesses communities characterized by a distinctive mix of species derived from Indo‐Pacific, Panamic, Peruvian and endemic source areas. The conservation significance of different regions is not reflected in counts of total species richness. The regions with the lowest overall fish species richness possess a temperate rather than tropical climate and highest levels of endemism.