The spatial and temporal distributions of the epiphytic diatom flora on Thalassia testudinum was described within the Florida Bay estuary and at one Atlantic site east of the Florida Keys over a 1-year period. Species of the genus Mastogloia dominated the epiphytic diatom flora (82 out of 332 total species). Nonmetric Multidimensional Scaling (NMDS) and Analysis of Similarity (ANOSIM) revealed four distinct spatial assemblages and two temporal assemblages. Eastern and western Florida Bay assemblages were identified within the estuary. The eastern diatom assemblage was characterized by high relative abundances of Brachysira aponina and Nitzschia liebetruthii, while the western assemblage was characterized by the abundance of Reimerothrix floridensis, particularly during summer. Two diverse and distinct marine assemblages, one located in the Gulf of Mexico along the western edge of Florida Bay and the other behind the Florida reef tract in the Atlantic Ocean, were also identified. Analysis of the spatial distribution of diatoms and water quality characteristics within Florida Bay suggest that these assemblages may be structured by salinity and nutrient availability, particularly P. The Gulf of Mexico and the western Florida Bay assemblages were associated with higher water column salinities and TP concentrations and lower DIN concentrations and TN:TP ratios relative to the eastern Florida Bay assemblage. The temporal variation in diatom assemblages was associated with water temperature, though temporal indicator species were few relative to the number of spatial indicators.