The hexaploid Asplenium cyprium Viane et Van den heede sp. nov. and its pentaploid hybrid, A. xtroodeum Viane et Van den heede hybr. nov., with A. ceterach are described as new taxa for Cyprus. Both are similar in gross morphology to diploid A. javorkeanum and to tetraplold A. ceterach, and can best be differentiated cytologically by counting chromosomes. If this is impossible (e.g., for herbarium material) the exospore and stomatal guard cell size also allow reliable identification. Isozyme analysis is used to prove that A. cyprium is different from the Madeiran hexaploid A. lolegnamense. Identical or very similar arrays of allozymes for A. javorkeanum, A. ceterach, and A. cyprium, confirm the autopolyploidy of A. ceterach and A. cyprium. Many of the heterozygous tetraploid and hexaploid plants exhibit unbalanced staining activity, implying tetrasomic and hexasomic inheritance. In contrast, five loci (Aat, Skdh, Pgi-2, Tpi-1 and Tpi-2) show fixed heterozygosity for the Madeiran A. lolegnamense and confirm its allohexaploid origin. The Pgi-2 zymograms indicate a multiple and local origin of autohexaploid A. cyprium. The observed patterns are consistent with those expected under the hypothesis that A. cyprium originated directly via hybridization between a normal and an unreduced gamete of A. ceterach. The fact that A. javorkeanum and A. xmantoniae were not found on the Island (110 plants were cytologically checked) forms an additional argument to adopt this hypothesis.