St. Augustinegrass (Stenotaphrum secundatum (Walter) Kuntze) is a warm season grass widely distributed in the southern United States and mainly used in residential and recreational lawns.   The University of Florida’s St. Augustinegrass germplasm collection is the most extensive and diverse germplasm source for this species in the US, being several-fold larger than the national collection. Characterization of the genetic diversity present in this important germplasm pool could aid in its effective use in cultivar improvement. The objective of this study was to characterize levels of genetic diversity present in the UF collection, and compare them with those of commercial cultivars and the national collection. For this purpose, 15 AFLP primer combinations were used to amplify a total of 805 polymorphic loci in 274 genotypes. Genetic similarity values among genotypes ranged from 0.12 to 0.98. Cluster analysis, using different methods, and principal coordinate analysis were performed. Genotypes were grouped into clusters mainly according to their ploidy level. Data generated from this investigation will be helpful when selecting parents for the development of breeding populations in this important turfgrass species.