Random Amplified Polymorphic DNA (RAPD) markers were used to measure the levels of genetic variation and patterns of the population structure within and among the five remaining populations of Ceratopteris pteridoides, an endangered aquatic fern in China. Fourteen RAPD primers amplified 101 reproducible bands, with 34 (33.66%) of them being polymorphic, indicating low levels of genetic diversity at the species level. The level of genetic diversity within the populations was considerably lower, with the percentage of polymorphic bands (PPB) ranging from 16.83% to 24.75%. AMOVA analysis revealed a low level of genetic variation (30.92%) among the populations. The UPGMA cluster of 72 samples detected that individuals from the same population did not form one distinct group, indicating high levels of gene flow between the populations. A Mantel test showed no significant relationship between genetic distance and geographic distance (r = 0.2166). Our results were similar to those obtained in an earlier ISSR analysis. Thus both RAPD and ISSR markers have comparable sensitivity, and could be employed to assess the partition of genetic diversity within and among populations. Several factors including clonal growth, inbreeding, high spore dispersal and the extensive hydrologic connectivity among populations which facilitate long-distance gene flow, might have played an important role in maintaining the genetic structure of the populations. In view of the genetic information currently available, we recommend establishing as many in situ conservation spots as possible and the cross transplanting of plants between populations in order to increase gene flow and preserve the genetic resources of the species.