Species boundaries and relationships were investigated in the Asplenium paleaceum (Aspleniaceae) species complex from eastern Australia, using AFLP fingerprinting and chloroplast trnL–trnF and rps4–trnS and nuclear pgiC DNA sequences. Phenetic analyses of AFLP data resulted in the following five distinct groups: (1) A. carnarvonense, (2) A. bicentenniale (including nearby collections originally identified as A. paleaceum), (3) A. paleaceum with both aborted and normal spores, (4) one population of putative tetraploid A. attenuatum var. indivisum from south Queensland, and (5) remaining octoploid A. attenuatum populations and several putative hybrids. Taxonomic revision of this complex will require morphological re-circumscription of the current species and recognition of a new species if these AFLP groups are taken to represent separate species. The chloroplast regions, morphology and pgiC together provide good evidence that an Asplenium of unconfirmed identity, A. sp. ‘Kroombit Tops’, is an allopolyploid with a species of the A. paleaceum chloroplast clade, probably A. paleaceum, and distantly related A. polyodon as parents. Further study is required to determine the complete ancestry of the other species of the A. paleaceum complex.