The uplands of the Reserve are predominantly higher elevation sand dunes of Pleistocene age, mixed with various organic sediments and soils. Much of these uplands are at a mean elevation of four feet, but a sandy ridge running roughly parallel with Shell Island road in a north-south direction is more than five and one-half feet in elevation. These sandy regions are intermediately to well-drained and, as a consequence, support typical xeric vegetational assemblages. The highest elevation in the Reserve is 22 feet above mean sea level.

An unusual upland feature of RBNERR and the region are shell mounds. These are mostly kitchen middens and refuse sites used by the aboriginal Calusa Indians. They are often prominent above the low-lying contiguous tidelands of the Reserve.

The lagoonal bays that comprise Rookery Bay, Dollar Bay and Johnson Bay are part of a larger interconnected system that once extended all the way up the western Florida coast to the vicinity of Tampa Bay. Many of these bays, which formed on the landward side of enclosing or barricading barrier islands, have filled in over time either through natural hydrographical processes or by human activities. Consequently, only ragged remnants exist north of Rookery Bay.

The barrier islands in the vicinity of Rookery Bay, have become coalesced into incipient (Marco Island) or actual (Naples area) headlands. However, the extensive mangrove-dominated ecosystem continued to flourish and to expand into these areas as well as the coastal mainland. It now forms a vast uninterrupted coastal ecosystem from south of Naples to the lower southeastern margin of Florida Bay in Everglades National Park.