The Sanford and Lake Eustis Railway was a historic railroad that ran from Sanford, Florida west to the town of Tavares on Lake Eustis, a distance of 28 miles. The line served as a branch of the Jacksonville, Tampa and Key West Railway, a Plant System railroad, that later became part of the Atlantic Coast Line Railroad.

The Jacksonville, Tampa and Key West Railway was a railroad and steamboat network in Florida at the end of the 19th century. Most of its lines became part of the Plant System in 1899 and the Atlantic Coast Line Railroad in 1902. The line remains in service today with a vast majority of it now being CSX Transportation's Sanford Subdivision.