The genetic diversity within and among 13 populations of the endangered aquatic fern Ceratopteris thalictroides from five regions of China was investigated using random amplification of polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers. The percentages of polymorphic bands were 61% for RAPD and 65% for ISSR. The genetic diversity revealed by RAPD and ISSR varied greatly among populations, with the percentages of polymorphic bands ranging from 15% to 39% for RAPD and from 20% to 39% for ISSR. Analysis of molecular variance showed that 36% (RAPD) and 34% (ISSR) of variability was partitioned among populations. The results indicate that C. thalictroides possesses an intermediate level of genetic diversity at the species level and a low level of genetic differentiation among populations. The Mantel test showed no significant correlation between genetic distance and geographical distance (RAPD, r = 0.53; ISSR, r = 0.18) and, similarly, a very poor fit between the two markers (r = 0.24). A number of causes, including inbreeding and clonal growth, may have led to the low genetic diversity within populations. A high gene flow via spore dispersal in an earlier period when C. thalictroides was widely distributed in China is a plausible reason for the low genetic differentiation among populations. Strategies for the conservation of the species in China are discussed. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, 657–671.