To examine the performance and information content of different marker systems, comparative assessment of populaiton genetic diversity was undertaken in nine populations of Athyrium distentifolium using nine genomic and 10 expressed sequence tag (EST) microsatellite (SSR) loci, and 265 amplified fragment length polymorphism (AFLP) loci from two primer combinations. In range-wide comparisons (European vs. North American populations), the EST-SSR loci showed more reliable amplification and produced more easily scoragle bands than genomic simple sequence repeates (SSRs). Genomic SSTs showed significantly higher levels of allelic diversity than EST-SSRs, but there was a significant correlation in the rank order of population diversities revealed by both marker types. When AFLPs, gneomic SSRs and EST-SSRs are considered, comparisons of different population diversity metrics/markers revealed a mixture of significant and nonsignificant rank-order correlations. However, no hard incongruence was detected (in no pairwise comparison of populations did different marker systems or metric detect opposingly significant different amounts of variation). Comparable population pairwise estimates of Fst were obtained for all marker types, but whilst absolute values for genomic and EST-SSRs were very similar (Fst = 0.355 and 0.342, respectively), differentiation was consistently higher for AFLPs in pairwise and global comparisons (global AFLP Fst - 0.496). The two AFLP primer combinations outperformed 18 SSR loci in assignment tests and discriminatory power in phenetic cluser analysis. The results from marker conparisons on A. distentifolium are discussed in the context of the few other studies on natural plant populations comparing microsatellite and AFLP variability.