The Big Cypress Swamp (BCS) is a large freshwater wetland system and drainage basin (640,000 ha) in southwest Florida and an important component of the Greater Everglades ecosystem. Despite its size and relationship to the Ever glades, the fish fauna of BCS has received little study. Documentation of its fish fauna is important to better understand this dynamic natural system and to monitor changes to the fish community, including the spread of non-indigenous species. To that end, we surveyed the ichthyofauna of freshwater habitats in Big Cypress National Preserve (BCNP), the largest and most intact wetland area (295,000 ha) remaining in BCS. Between October 2002 and May 2004, we recorded 63 fish species from freshwater habitats in BCNP, including 9 non-indigenous species. Species richness was greatest in permanent freshwater habitats and lowest in shallow temporary wetlands and seasonally fresh coastal marshes. The most speciose families were the native Centrarchidae (8 spp.) and the non-native Cichlidae (6 spp.), whereas the most abundant and widely distributed species were members of Cyprinodontidae, Fundulidae, and Poecilidae. Similar to other coastal drainages of southern Florida, BCNP has a relatively high occurrence of euryhaline species (28 spp.).