The Asplenium obovatum group consists of diploid and tetraploid taxa; the origin of the tetraploid A. obovatum subsp. lanceolatum was previously considered to have occurred via autopolyploidy, involving one of the diploids of the group, either A. obovatum subsp. obovatum var. obovatum or var. protobillotii. To test this hypothesis, electrophoretic analyses of eight enzyme systems encoded by fourteen putative loci and cytological studies of the artificial hybrid between both diploid varieties were conducted. Alleles of the loci Lap-1, Mdh-2, Mdh-3, Pgm-1, Pgm-1', and 6Pgd-1 emerged as genetic markers for the diploids and were present in an additive pattern in most of the analyzed individuals of the tetraploid. Cytological results indicated a high degree of genomic homology between the diploids. These results indicated that the tetraploid behaves as a segmental allopolyploid. Our results showed that both diploids were involved in the origin of the tetraploid. We propose the new combination Asplenium obovatum subsp. protobillotii for one of the diploids.