Thalassia testudinum (Banks ex König) is an important primary producer in nearshore tropical marine ecosystems. In the past several years, mass mortality of T. testudinum populations within the Florida Bay estuary has highlighted the need to know more about levels of clonal variation and the spatial arrangement of genets within populations of this species. Historically, most of the reproduction within this species was thought to be through clonal growth, and populations have been assumed to exhibit little genetic diversity. We used DNA fingerprinting techniques and spatially hierarchical sampling to search for genetically distinct plants within T. testudinum meadows in Rabbit Key Basin, Florida Bay, USA. We detected 22 genetically distinct individuals out of 74 samples. Multiple genetic individuals were detected over small spatial scales (<0.25 m). Analysis of molecular variance (AMOVA) detected significant levels of variation at both the largest and smallest spatial scales studied. This result suggested that genets of this species generally do not grow in discreet monoclonal patches and that most genets are not large enough to span the entire basin (∼5 km). Further, analysis of genetic distances between phenotypes suggests a pattern of isolation by distance, with individuals that were nearer in space being more genetically similar. GIS analysis of seagrass density over the period from 1985 to 1994, combined with the clonal diversity data presented here, suggest seedlings may play a significant role in colonization of bare patches. These results imply that the role of sexual reproduction in population structure within this species is greater than previously thought.