Amplified fragment length polymorphisms (AFLPs) were used to characterize the genetic diversity within and among natural populations of Sticherus flabellatus. Eight populations within the Sydney region of New South Wales, Australia were surveyed using 11 primer combinations. A total of 1108 reproducible bands were detected of which 469 (42%) were polymorphic. FST estimates averaged over all polymorphic loci indicated that significant genomic differentiation occurs among populations (average = 0.783). Genetic diversity within populations was assessed according to average heterozygosity (H) and percentage polymorphic loci (P) per population. Within-population diversity ranged from H = 0.12 and P = 33.69 to H = 0.04 and P = 15.99. Analysis of genetic similarity among populations suggested that the eight populations studied fall into two groups of four populations, based on population size and the condition of the habitat. Phenetic analysis (amova) indicated that genetic variation is greater among populations (74.34%) than within populations (25.66%). These findings suggest that the breeding system of S. flabellatus is predominantly inbreeding, with genetic diversity maintained by occasional outcrossing in larger populations. The results presented in this study could provide evidence to support the proposal to protect natural stands of S. flabellatus, which has implications for the Australian horticulture industry.