The breeding system of Fossombronia foveolata and genetic differentiation in the F. foveolata species complex were studied using Random Amplified Polymorphic DNA (RAPD) markers. Polymorphism values for cloned siblings were not significantly different from those obtained from amplification of identical DNA samples. These results support the hypothesis that sporophytes display high levels of homozygosity. Nevertheless, low levels of genetic differentiation were detected between F. foveolata populations from spatially separated sites in southern Illinois. Monomorphism among siblings, low levels of polymorphism among southern Illinois populations, sporophyte production in clonal cultures maintained in an environmental chamber, growth of cultured clonal mats to sizes comparable to field populations, and short sperm and spore dispersal distances suggest that F. foveolata populations are probably established from single founder events and are maintained through inbreeding and vegetative reproduction. Degree of genetic relatedness among F. foveolata from Illinois, F. foveolata from Texas, F. cristula from Indiana, F. japonica from Japan, F. lamellata from Arkansas, F. porphyrorhiza from Brazil, F. pusilla from California, and F. texana from Texas were also estimated from RAPD data. UPGMA cluster analyses of several different similarity matrices resolved the following three species groups 1) F. foveolata, F. lamellata, F. cristula group; 2) F. japonica, F. porphyrorhiza, F. pusilla group; and 3) F. texana. These groupings are consistent with those based on analyses of morphological characters, as well as other molecular data sets.