The Florida Reef Tract (FRT) extends from the tropical Caribbean up the southeast coast of Florida into a temperate environment where tropical reef assemblages diminish with increasing latitude. This study used data from a three-year comprehensive fishery-independent survey to quantify reef fish spatial distribution along the Southeast FRT and define where the assemblage shifts from tropical to temperate. A total of 1,676 reef fish visual census samples were conducted to assess the populations on a stratified-random selection of sites of marine hardbottom habitats between the Miami River and St. Lucie inlet. Multivariate analyses were used to investigate differences in assemblages among sites. Depth (m), general habitat (reef or hardbottom), and slope (high or low) strata were examined to explain the dissimilarities between assemblages. A general trend of cold-tolerant temperate fish dominated the northern assemblages and more tropical species dominated further south. Seven reef fish assemblage biogeographic regions were determined. In shallow habitats the data clustered in three spatial regions: One south of Hillsboro inlet, one in Northern Palm Beach south of Lake Worth inlet, and one north of Lake Worth inlet. The assemblage in deep habitats mainly split in close proximity to the Bahamas Fracture Zone south of Lake Worth Inlet. The presence of reef habitat aided in splitting the southern assemblage regions from the northern all-hardbottom assemblage regions in both the shallow and deep habitats. Substrate relief was significantly correlated with the differences in the northernmost deep assemblages but did not appear to affect the remainder of the shallow and deep assemblages. This bioregional study creates a baseline assessment of reef fish assemblages of the Southeast FRT for future analyses.