The 4,053 acre refuge includes Pinckney Island, Corn Island, Big and Little Harry Islands, Buzzard Island and numerous small hammocks. Pinckney is the largest of the islands and the only one open to public use. Nearly 67% of the refuge consists of salt marsh and tidal creeks. A wide variety of land types are found on Pinckney Island alone: salt marsh, forestland, brushland, fallow field and freshwater ponds. In combination, these habitats support a diversity of bird and plant life. Wildlife commonly observed on Pinckney Island include waterfowl, shorebirds, wading birds, raptors, neo-tropical migrants, white-tailed deer and American alligators, with large concentrations of white ibis, herons, and egrets.

Forest thinning allows sunlight to reach the forest floor and promotes the growth of valuable wildlife foods such as blackberry bushes. As competition for existing resources diminishes, remaining trees grow more vigorously and forest stands produce better quality trees.

To maintain wildlife openings, areas are periodically mowed and disked. This interrupts the natural succession of plant communities toward their climax stage, and prevents beneficial annual and perennial grasses from being replaced with woody shrubs or trees. "Edge species" which frequent the borders between two different succession stage areas prefer these open spaces. In addition, some species may require an assortment of habitats to fulfill their entire life cycle, therefore a mosaic of different habitats is required. The freshwater ponds provide loafing and resting areas for migrating waterfowl during fall.