A scientifically defensible methodology for identifying areas of high biodiversity in aquatic environments is presented. Areas of high biodiversity or Aquatic Biodiversity Investment Areas are identified using a technique referred to as Habitat Supply Analysis. This technique uses the microhabitat features of an ecosystem in conjunction with information on the microhabitat preferences of fish to calculate the suitability of an area to fish. The method is structured so that the suitability of habitat to lifestages of fish, species of fish, groups of fish and fish assemblages can be evaluated. The methodology recognises that to some degree all areas within an aquatic system contribute to the maintenance of biodiversity. As such, a classification scheme is proposed to evaluate the potential versus the actual contribution of an area to the maintenance of biodiversity in an ecosystem. This classification scheme is designed to help prioritise habitat restoration and preservation efforts. Prototype evaluations of the methodology for identifying and classifying Aquatic Biodiversity Investment Areas are presented.